FUNCTIONAL AND PHARMACOLOGICAL ANALYSIS OF INSECT SODIUM
CHANNELS IN XENOPUS OOCYTES
By
Lingxin Wang

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Entomology - Doctor of Philosophy
2013

ABSTRACT
FUNCTIONAL AND PHARMACOLOGICAL ANALYSIS OF INSECT SODIUM
CHANNELS IN XENOPUS OOCYTES
By
Lingxin Wang
Voltage-gated sodium channels are responsible for electrical signaling in the nervous
system and other excitable cells. Insects take advantage of extensive alternative splicing and
RNA editing of a single gene to achieve functional diversity of sodium channels. TipE and four
TipE-homologous genes (TEH1-4) in Drosophila melanogaster are thought to encode auxiliary
subunits of sodium channels. However, limited information is available on how TipE and TEH
proteins modulate the gating of sodium channels. Sodium channels are the primary target site of
pyrethroid insecticides. Due to intensive use of pyrethroids, resistance has become a serious
problem for pest control. A major mechanism of pyrethroid resistance, known as knockdown
resistance (kdr), is caused by mutations in the sodium channel. In addition to kdr mutations,
naturally occurring sodium channel variants exhibit different sensitivities to pyrethroids.
The objectives of my Ph.D. dissertation are: (1) to investigate the effects of TipE and
TEH1-4 on the gating properties and sensitivity of sodium channel variants (DmNav) from D.
melanogaster to pyrethroid insecticides; (2) to explore the involvement of RNA editing in
regulating channel gating and sensitivity to pyrethroids; and (3) to determine whether a naturally
occurring mutation contributes to pyrethroid resistance in mosquitoes.
For the first objective, TipE or TEH1-4 was co-expressed individually with three
different DmNav variants in Xenopus oocytes, and gating properties were determined by twoelectrode voltage clamp. TEH1 and TEH4 induce hyperpolarizing and depolarizing shifts,

respectively, in the voltage-dependence of activation, fast inactivation, and slow inactivation;
TEH2 does not modulate the gating properties; and TipE modulates only the voltage-dependence
of fast inactivation of one variant. TEH3 enhances an outward current of an endogenous
channel(s) in Xenopus oocytes, which prevented further analysis of its effect on the sodium
channel variants. For the second objective, the role of RNA editing in gating and channel
sensitivity to pyrethroids was investigated by individually introducing four RNA editing sites
into an unedited variant DmNav26. One RNA editing event, N1587S, causes a hyperpolarizing
shift in the voltage-dependence of slow inactivation of the DmNav26 channel. Another RNA
editing event, Q1296R, enhances the sensitivity of DmNav 26 channels to a pyrethroid
insecticide, deltamethrin. For the third objective, whether a naturally occurring mutation
N1575Y, detected from pyrethroid-resistant populations of Anopheles gambiae mosquitoes,
contributes to resistance to pyrethroids was investigated. I found that the N1575Y mutation alone
does not alter the gating or sensitivity of a mosquito sodium channel to deltamethrin. However,
the N1575Y mutation augmented channel sensitivity to deltamethrin induced by two kdr
mutations L1014F/S located in pyrethroid receptor site 2, but not by a kdr mutation, V1016G,
located in pyrethroid receptor site 1. These results suggest that the N1575Y mutation functions
as an enhancer of site-2 kdr mutations.
In conclusion, my research shows critical roles of TipE/TEH proteins and RNA editing in
the regulation of insect sodium channel function. Furthermore, identification of both the role of
RNA editing in regulating channel sensitivity to pyrethroids and the N1575Y mutation as an
enhancer for pyrethroid resistance in mosquitoes provide new insights into the molecular
mechanisms of pyrethroid resistance.

ACKNOWLEDGEMENTS

Completing my Ph.D. degree is probably the most challenging, exciting, and memorable
activity of my life so far. During my four years of study at Michigan State University, I was
supported and assisted by many people. I could not have gotten to this point without their help.
First of all, I want to give my gratitude to my mentor, Dr. Ke Dong, who supported me
the most during my study here. Her patience, encouragement, and profound knowledge helped
me overcome many obstacles during my Ph.D. study. Whenever I had questions, she was there to
offer me guidance. She has been a strong and supportive advisor, but she always gave me the
freedom to pursue my research interests. She has been and will always be a standard model for
me to follow as a scientist in my future career.
Secondly, I want to give my great thanks to my dear committee members, Dr. Suzanne
Thiem, Dr. Zachary Huang, and Dr. Peter Cobbett for their support, guidance and useful
suggestions. Dr. Thiem always encouraged me to be confident and also informed me a lot of
American culture and customs. Dr. Huang was always very patient in answering my questions
and invited me to some social activities. Dr. Cobbett gave me helpful suggestions on how to
prepare my comprehensive exam and proposal defense, including how to search for the projectrelated literature online.
Thirdly, members of our lab, both past and present, deserve my sincere thanks for their
friendship and assistance. Dr. Yuzhe Du taught me how to do electrophysiological experiments
and data analysis. From Ms. Yoshiko Nomura, I learned how to conduct cloning and sitedirected mutagenesis. Dr. Frank Rinkevich shared his own experience on how to write a thesis.
Mr. Peng Xu spoke with me on his experiences in job hunting. I also want to thank former lab

iv

members Dr. Tianxiang Zhang, Dr. Zhaonong Hu, and Dr. Rong Gao. Dr. Zhang taught me how
to run insect behavioral assays and insecticide bioassays. Dr. Hu and Dr. Gao provided
unpublished data which were the foundation for Chapter X of my dissertation. I also want to
thank our undergraduate lab assistant, Mr. Joshua Tolinski, for rearing Xenopus frogs which I
used in my research.
Last but not least, I am forever indebted to my parents and my wife for their
understanding, endless patience, and encouragement. I also want to give my appreciation to my
parents-in-law, who have always had a strong faith in me and have encouraged me to do the
things that I like the most. I also want to thank my sisters for taking care of my parents while I
am in the U.S.

v

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………………..ix
LIST OF FIGURES………………………………………………………………………………x
CHAPTER 1
GENERAL INTRODUCTION……………………………………………………………………1
1.1. Sodium channels……………………………………………………………………………...2
1.1.1. History and Structure of sodium channels………………………………………………..2
1.1.2. Sodium channel voltage-dependent gating properties……………………………………3
1.1.3. Mammalian sodium channel α subunits…………………………………………………..4
1.1.4. Mammalian sodium channel β subunits…………………………………………………..5
1.1.5. Insect sodium channels…………………………………………………………………...7
1.1.6. Insect sodium channel auxiliary subunits………………………………………………...7
1.2. Alternative splicing and RNA editing………………………………………………………...9
1.2.1. Alternative splicing……………………………………………………………………….9
1.2.2. RAN editing……………………………………………………………………..............10
1.3. Pyrethroids…………………………………………………………………………………..11
1.3.1. Pyrethrum and pyrethroids………………………………………………………………11
1.3.2. Type I and type II pyrethroids…………………………………………………………..12
1.3.3. The mode of action of pyrethroids………………………………………………………13
1.3.4. Knockdown resistance (kdr)…………………………………………………………….13
1.3.5. The pyrethroid receptor sites……………………………………………………………14
1.4. Differential sensitivity of sodium channel variants to pyrethroids and Av3………………..15
CHAPTER 2
DIFFERENTIAL EFFECTS OF TIPE AND A TIPE-HOMOLOGOUS PROTEIN ON
MODULATION OF GATING PROPERTIES OF SODIUM CHANNELS FROM
DROSOPHILA MELANOGASTER……………………………………………………............25
2.1. Abstract………………………………………………………………………………….......26
2.2. Introduction………………………………………………………………………………….27
2.3. Materials and Methods………………………………………………………………………30
2.3.1. Xenopus oocyte expression system……………………………………………………...30
2.3.2. Electrophysiological recording and analysis……………………………………………30
2.3.3. Measure of sodium channel sensitivity to deltamethrin………………………………...33
2.3.4. Chemicals………………………………………………………………………………..33
2.3.5. Statistical analysis……………………………………………………………………….34
2.4. Results……………………………………………………………………………………….34
2.4.1. TipE and TEH1 increased the peak sodium current and accelerated the current decay of
all three DmNav variants………………………………………………………………………...34
2.4.2. Differential effects of TEH1 and TipE on the voltage-dependence of activation and fast
inactivation……………………………………………………………………………………….35
2.4.3. Effect of TipE and TEH1 on the sensitivity of DmNav9-1 channels to deltamethrin…..35
vi

2.4.4. Co-expression of TEH1 with DmNav9-1 significantly enhanced entry into and stability
of the fast inactivated state……………………………………………………………………….37
2.4.5. Co-expression of TEH1 with DmNav9-1 inhibited recovery from slow inactivation…..37
2.5. Discussion…..........................................................................................................................38
CHAPTER 3
DISTINCT MODULATING EFFECTS OF TIPE-HOMOLGOUS PROTEINS 2 AND 4 ON
DROSOPHILA SODIUM CHANNEL SPLICE VARIANTS…………………………………..56
3.1. Abstract……………………………………………………………………………...............57
3.2. Introduction………………………………………………………………………………….58
3.3. Materials and Methrods……………………………………………………………………..61
3.3.1. Xenopus oocyte expression system……………………………………………………...61
3.3.2. Electrophysiological recording and analysis……………………………………………61
3.3.3. Statistical analysis……………………………………………………………………….64
3.4. Results……………………………………………………………………………….............64
3.4.1. TEH2 increased the amplitude of peak sodium current of DmNav channels…………...64
3.4.2. TEH4 inhibited sodium current decay and increased the persistent current of DmNav
channels………………………………………………………………………………..................65
3.4.3. TEH4 altered the voltage-dependence of activation, fast inactivation, and slow
inactivation of DmNav9-1, DmNav22, and DmNav26…………………………………………..66
3.4.4. TEH4 altered the kinetics of fast inactivation of DmNav9-1 channels…………………66
3.5. Discussion…………………………………………………………………………............67
CHAPTER 4
THE N1575Y MUTATION IN THE SODIUM CHANNEL OF ANOPHELES GAMBIAE
SYNER GI ZES THE EFFECT OF L101 4F/ S M UTATI ONS ON PY R ETHROI D
RESISTANCE…………………………………………………………………………...............91
4.1. Abstract……………………………………………………………………………………...92
4.2. Introduction……………………………………………………………….…………………93
4.3. Materials and Methods………………………………………………………………………95
4.3.1. Site-directed mutagenesis………………………………………………………….........95
4.3.2. Expression of AaNav sodium channels in Xenopus oocytes……………………………95
4.3.3. Electrophysiological recording and analysis……………………………………………95
4.3.4. Measurement of tail currents induced by pyrethroids…………………………………...96
4.3.5. Chemicals………………………………………………………………………………..97
4.3.6. Statistical analysis……………………………………………………………………….97
4.4. Results………………………………………………………………………………….........98
4.4.1. N1575Y did not alter the current amplitude or gating properties of the AaNav1-1
channel……………………………...............................................................................................98
4.4.2. N1575Y had no effect on AaNav1-1 channel sensitivity to pyrethroids but enhanced
L1014F-mediated resistance to pyrethroids……………………………………………………...99
4.4.3. N1575Y enhanced the L1014S-mediated resistance to pyrethroids…………………….99
4.4.4. N1575Y has no effect on the V1016G-mediated resistance to pyrethroids……………100
vii

4.5. Discussion…………………………………………………………………….....................100
CHAPTER 5
A-TO-I EDITING IN THE DROSOPHILA SODIUM CHANNELS MODIFIES THE GATING
AND SENSITIVITY OF SODIUM CHANNELS TO DELTAMETHRIN AND AV3……….113
5.1. Abstract………………………………………………………………………….................114
5.2. Introduction………………………………………………………………………………...115
5.3. Materials and Methods……………………………………………………………………..117
5.3.1. Site-directed mutagenesis………………………………………………………….......117
5.3.2. Expression of sodium channels in Xenopus oocytes…………………………………...117
5.3.3. Electrophysiological recording and analysis…………………………………………..117
5.3.4. Measurement of tail currents induced by pyrethroids………………………………….119
5.3.5. Measurement of sodium channel sensitivity to Av3…………………………………...119
5.3.6. Chemicals………………………………………………………………………………120
5.3.7. Statistical analysis……………………………………………………………………...120
5.4. Results……………………………………………………………………………………...120
5.4.1. Substituting exon l for exon k significantly increased the persistent sodium current and
sensitivity of DmNav26 channels to deltamethrin……………………………………………...120
5.4.2. Three amino acid changes unique to DmNav26 did not confer channel resistance to
deltamethrin and Av3…………………………………………………………………………...121
5.4.3. N1587S in the linker connecting somains III and IV caused a hyperpolarizing shift in the
voltage-dependence of slow inactivation……………………………………………………….122
5.4.4. Q1296R substitution increased DmNav26 channel sensitivity to deltamethrin and
Av3………………………………………………………………………………………...........123
5.5. Discussion………………………………………………………………………………..123
CHAPTER 6
FUTURE DIRECTION………………………………………………………………………...148
BIBLIOGRAPHY………………………………………………………………………………151

viii

LIST OF TABLES

Table 1-1. Tissue distribution of nine mammalian voltage-gated sodium channel α subunits
(Goldin 2000, Yu et al., 2003)…………………………………………………………………...16
Table 2-1. Gating properties of DmNav variants with or without TipE or TEH1……………….43
Table 2-2. Development of slow inactivation of DmNav9-1 with or without TipE or TEH1…...44
Table 2-3. Recovery from slow inactivation of DmNav9-1 with or without TipE or TEH1…….44
Table 3-1. TEH1, TEH2 and TEH4 modulated voltage-dependence of activation, fast
inactivation, and slow inactivation of DmNav9-1, DmNav26, and DmNav22 expressed in
Xenopus oocytes. ………………………………………………………………………………...73
Table 4-1. Voltage-dependence of activation and fast inactivation of AaNav1-1 and its mutant
channels…………………………………………………………………………………............104
Table 5-1. Voltage-dependence of activation, fast inactivation, and slow inactivation of
DmNav26 and its mutants………………………………………………………………............128
Table 5-2. Time constant of tail current decay induced by deltamethrin………………............129

ix

LIST OF FIGURES

Figure 1-1. Topology of the voltage-gated sodium channel α subunit. The α subunit is composed
of four homologous domains (I-IV), each of which contains six transmembrane segments (S1S6). Positively charged amino acid residues in S4s are represented as +, which serve as voltage
sensors. The S5 and S6 segments and the P-region between them together form the inner pore of
the sodium channel. Amino acid residues EEDD and DEKA in the linker connecting S5 and S6
in each domain form two rings that are essential for ion selectivity. The black dot in the linker
connecting domains III and IV represents the fast inactivation particle, IFMT (isoleucine,
phenylalanine, methionine, threonine), which is thought to occlude the pore. (Modified from
Catterall, 2000)…………………………………………………………………………………..17
Figure 1-2. Structure of mammalian sodium channel β subunits and insect sodium channel
auxiliary subunits. (A) Structure of β subunits. Mammalian sodium channel β subunits are small
transmembrane proteins that possess an extracellular immunoglogulin (Ig) domain, a single
transmembrane segment, and a short intracellular C-terminal domain. (B) Structure of
TipE/TEH1-4. Insect sodium channel auxiliary subunit has two transmembrane segments
connected by one extracellular linker and both N- and C-terminus are intracellular………….18
Figure 1-3. Distribution of alternative exons on DmNav transcripts. A schematic diagram of the
DmNav sodium channel labeled with names and locations of 11 alternative exons indentified in
DmNav transcripts. Exons a, b, e, f, h, I, and j are optional exons. Exons d/c and l/k are two pairs
of mutually exclusive exons. (Modified from Olson et al., 2008)……………………………….19
Figure 1-4. Localization of 20 A-to-I RNA editing sites in the Drosophila sodium channel. From
our collaborators in Zhejiang University in China and Yale University………………………...20
Figure 1-5. Chemical structure of type I and type II pyrethroids. Cyano group in type II
pyrethroids is labeled as *………………………………………………………………………..21
Figure 1-6. Differential sensitivity of DmNav sodium channel variants to Av3. Sodium channel
variants were transiently expressed individually in Xenopus oocytes. Sodium channel sensitivity
to 250 nM of Av3 was measured by normalizing peak of non-inactivating current at end of 20 ms
d e p o l a r i z a t i o n t o p e a k o f t r a n s i e n t s o d i u m c u r r e n t . ( R e s u l t s f r om D r . R o n g
Gao)…………………………………………………………………………...………………...23
Figure 1-7. Differential sensitivity of DmNav sodium channel variants to deltamethrin. Sodium
channel variants were transiently expressed individually in Xenopus oocytes. Percentages of
channels modified by 1 µM pyrethroids was calculated using the equation
M={[Itail/(Eh−ENa)]/[INa/(Et−ENa)]}×100 (Tatebayashi and Narahashi, 1994),where Itail is the
maximal tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is
the reversal potential for sodium current determined from the current–voltage curve, INa is the
x

amplitude of the peak current during depolarization before pyrethroid exposure, and Et is the
potential of step depolarization. (Results from Dr. Zhaonong Hu)…………………….………..24
Figure 2-1. Modulatory effects of TipE and TEH1 on peak sodium currents of DmNav9-1,
DmNav22, or DmNav26 channels. (A) Representative traces of maximum peak sodium current
recorded as introduced bellow from oocytes expressing DmNav9-1, DmNav9-1+TipE, and
DmNav9-1+TEH1 sodium channels. Note that the sodium current of the DmNav9-1 channel
possesses a non-inactivating component, known as persistent current (10% of the maximal
transient peak current). TipE and TEH1 did not enhance the persistent current. (B) Both TipE
and TEH1 significantly increased peak sodium currents of all three Para sodium variants tested:
DmNav9-1, DmNav26, and DmNav22, but there was no significant difference between the
effects of TipE and TEH1. Sodium currents were recorded 48 hours after cRNA injection.
Sodium currents were recorded by a step depolarization to from -80 to 65 mV in 5mV
increments with a holding potential of -120 mV. Data are presented as mean ± SEM for 12-15
oocytes. * indicates a significant difference compared to peak of DmNav channel only using oneway ANOVA with Scheffe’s post hoc analysis (p < 0.05)……………………………45
Figure 2-2. Enhancement of sodium current decay by co-expression of TipE or TEH1 with
DmNav channel variants. Sodium current decay in DmNav9-1, DmNav22, or DmNav26 coexpressed with TipE (A, C, and E respectively) or TEH1 (B, D, and F respectively). The decay
of sodium current was fitted by a single exponential to generate time constants of current decay
(τdecay). Each data point represents mean ± SEM for 12-20 oocytes. * indicates a significant
difference compared to that of DmNav channel only (p < 0.05)………………………………47
Figure 2-3. Effects of co-expression of TipE or TEH1 on the voltage-dependence of activation
and fast inactivation of DmNav9-1 channels. (A) Voltage-dependence of activation. (B) Voltagedependence of fast inactivation. Data were fitted with a two-state Boltzmann equation and fitting
parameters are shown in Table 2-1. Data points are shown as mean ± SEM. Recording protocols
are indicated and the details of the protocols and data analysis are described in the Materials and
Methods………………………………………….……………………………………………….50
Figure 2-4. Sensitivity of DmNav9-1 to deltamethrin is modulated by co-expression of TEH1.
(A) A representative tail current induced by 1 µM deltamethrin. (B) Percentage of channel
modification by deltamethrin (multiple-pulse test). Tail currents were elicited by a 66.7-Hz train
of 100 5-ms depolarization from -120 to 0 mV. (C) Co-expression of TEH1 significantly reduced
the stability of DmNav9-1 peak sodium current under repeated conditioning depolarizations.
Sodium currents were recorded during 20-ms step depolarizations from –120 mV to -10 mV after
0-100 conditioning pulses (5-ms pulses from -120 mV to 0 mV at 66.7 Hz). (D) Percentage of
channel modification by deltamethrin (single-pulse test). Tail currents were elicited by a 500 ms
depolarization from -120 mV to 0 mV. All data are shown as mean ± SEM for 9-15 oocytes. *
indicates significant difference compared to the DmNav9-1 channel using one-way ANOVA
with Scheffe’s post hoc analysis (p < 0.05)…………………………………………………….51
xi

Figure 2-5. Effects of co-expression of TipE or TEH1 on entry into or recovery from fast
inactivation of DmNav9-1 channels. (A) Time course of development of fast inactivation with a
pre-pulse of -45 mV. (B). τ values of development of fast inactivation under different pre-pulse
potentials. τ values were determined by fitting time course of the development of fast
inactivation with a single exponential decay (n ≥ 12). (C). Recovery from fast inactivation with a
repolarizing potential of -70 mV. (D). τ values of recovery from fast inactivation at different
repolarizing voltages. τ values were calculated by fitting recovery from fast inactivation data
from different repolarizing voltages by a single exponential function10). Recording
≥ (n
protocols are indicated and the details of the protocols and data analysis are described in the
Materials and Methods…………………………………………………………………………...53
Figure 2-6. Co-expression of TEH1 inhibits recovery from slow inactivation of DmNav9-1
channels. (A) Voltage-dependence of slow inactivation. (B) Time course of development of
slow-inactivation. Development of slow inactivation was fitted by an exponential decay and the
parameters are summarized in Table 2-3. (C) Time course of recovery from slow-inactivation.
Recovery from slow inactivation was fitted by a double exponential function and the parameters
are summarized in Table 2. Recording protocols are indicated and the details of the protocols and
data analysis are described in the Materials and Methods……………………...………………..55
Figure 3-1. Effects of TEH2 and TEH4 on peak sodium currents of DmNav channels. (A)
Representative traces of maximum peak sodium currents recorded as introduced bellow from
oocytes expressing DmNav9-1, DmNav9-1 + TEH2, and DmNav9-1 + TEH4 channels. (B)
TEH2 and TEH4 differentially modulate peak sodium current of DmNav9-1, DmNav22 and
DmNav26 in Xenopus oocytes. Sodium currents were recorded 48 hours after cRNA injection.
Sodium currents were recorded by a step depolarization to -80 to 65 mV in 5mV increments
from a holding potential of -120 mV. Data are presented as mean ± SEM for 10-18 oocytes. *
indicates a significant difference compared to peak current of DmNav9-1 channel using a oneway ANOVA with Scheffe’s post hoc analysis (p < 0.05)…………………………....…………74
Figure 3-2. Expression of TEH3 alone in Xenopus oocytes induced a niflumic acid sensitive
outward current. Outward current was recorded by 40 msec depolarization from -100 mV to 130
mV with a holding potential of -60 mV. The outward current was recorded three days after
injection. (A) & (B) Outward current from oocyte injected with water before and 10 min after
niflumic acid application, respectively. (C) & (D) Outward current from TEH3 expressed oocyte
before and 10 min after niflumic acid application, respectively. (E) Amplitude of outward current
at different depolarizing voltages ranging from -100 mV to 130 mV without niflumic acid
application. (F) Amplitude of outward current from oocytes with or without TEH3 injection at
the depolarizing voltage of 130 mV before and after niflumic acid application. Data are shown as
mean ± SEM for 6-11 oocytes in E and F. * indicates significant difference using one-way
ANOVA with Scheffe’s post hoc analysis (p < 0.05)…………………………………………..76
Figure 3-3. TEH4, but not TEH2, inhibited the current decay of DmNav channels. (A) & (B) &
(C) TEH2 had no effect on sodium current decay of any of the three DmNav channels. (D) & (E)
xii

& (F) TEH4 inhibited sodium channel decay of all three DmNav channels. The decay of sodium
current was fitted by a single exponential to generate the time constants of current decay (τdecay).
Data are presented as mean ± SEM for 10-20 oocytes. * indicates a significant difference
compared to that of DmNav channels only (p < 0.05)………………………………………….79
Figure 3-4. Co-expression of TEH4 increased persistent sodium current of DmNav channels
expressed in Xenopus oocytes. (A) Representative peak current traces of DmNav9-1, or coexpression with TipE, TEH1-2, and TEH4. (B), (C), & (D) Modulating effects of TipE or TEH
proteins on persistent current of DmNav9-1, DmNav22, or DmNav26 channels, respectively. All
data are shown as mean ± SEM for 9-18 oocytes. * indicates significant difference compared to
the DmNav channels using one-way ANOVA with Scheffe’s post hoc analysis (p < 0.05).……82
Figure 3-5. Effects of TEH4 on the persistent current of DmNav9-1 channel under ramp protocol
test. (A) Representative traces of persistent current. (B) Normalized persistent sodium current at
different voltages. Amplitude of persistent currents was normalized to the amplitude of peak of
transient sodium current. All data are shown as mean ± SEM for 9-12 oocytes. * indicates
significant difference compared to the DmNa v9-1 channel using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05)……………………………...............................................84
Figure 3-6. Effects of co-expression of TEH2 and TEH4 on the voltage-dependence of
activation, fast inactivation, and slow inactivation of DmNa v 9-1 channels. (A) Voltagedependences of activation. (B) Voltage-dependence of fast inactivation. (C) Voltage-dependence
of slow inactivation. Data were fitted with a two-state Boltzmann equation and fitting parameters
are shown in Table 3-1. Data points are shown as mean ± SEM. Recording protocols are
indicated and the details of the protocols and data analysis are described in Materials and
Methods ………………………………………………………………………………………..85
Figure 3-7. Effects of co-expression of TEH2 or TEH4 on entry into or recovery from fast
inactivation of DmNav9-1 channels. (A) Time course of development of fast inactivation with a
pre-pulse of -45 mV. (B) τ values of development of fast inactivation under different pre-pulse
voltages. τ values were determined by fitting time course of the development of fast inactivation
with a single exponential decay (n > 10). (C) Recovery from fast inactivation with a repolarizing
voltage of -70 mV. (D) τ values of recovery from fast inactivation at different repolarizing
voltages. τ values were calculated by fitting recovery from fast inactivation data from different
repolarizing voltages by a single exponential function (n > 9)…………………………….......87
Figure 3-8. Neither TEH2 nor TEH4 altered the kinetics of slow inactivation of DmNav9-1
channels. (A) Neither TEH2 nor TEH4 altered the peak stability of DmNav9-1 channels; (B)
Development of slow inactivation or (C) Recovery from slow inactivation. Protocols for testing
peak stability, development of slow inactivation, and recovery from slow inactivation are
described in Materials and Methods…………………………………………………..................89
xiii

Figure 4-1. The positions of the pyrethroid resistance-associated mutations relevant to this study.
The sodium channel transmembrane protein consists of four homologous domains (I-IV), each
formed by six transmembrane segments (S1-S6) connected by intracellular and extracellular
loops. Residue positions correspond to the housefly sodium channel (GenBank accession
number: AAB47604 and AAB47605). Positions in parentheses correspond to AaNav sodium
channel (GenBank accession number: EU399181). L1014F/S mutations were detected in many
pyrethroid resistant populations in Anopheles gambiae (Rinkevich et al. 2013), and N1575Y was
detected in West and Central Africa populations of Anopheles gambiae (Jones et al., 2012). The
V1016G mutation was found in Aedes aegypti in many regions of the world (Rinkevich et al.,
2013). The entire amino acid sequence of the linker connecting domains III and IV is shown
below the topology diagram. The MFMT motif that is critical for fast inactivation is boxed. The
N1575Y mutation is marked in bold. A previously confirmed pyrethroid-sensing residue,
L1596P, in this linker is also shown in bold…...……………………………………….............105
Figure 4-2. Sodium currents from AaNav1-1 and mutant sodium channels. (A) Representative
current traces. The sodium currents were elicited by a 20-ms depolarization to 0 mV from the
holding potential of -120 mV. (B) Sodium current decay. Time constant of sodium current decay
was calculated by fitting the decaying part of current traces, elicited by a series of depolarizing
voltages, with a single exponential function ………………………………………………….106
Figure 4-3. Voltage dependences of activation and inactivation of AaNav1-1 and mutant sodium
channels. (A) & (B) Voltage-dependence of activation. (C) & (D) Voltage-dependence of
inactivation. Data analysis and the protocols for the voltage-dependence of activation and
inactivation are described in Materials and Methods. Symbols represent means, and error bars
indicate SEM values ……………………………………………………………………........107
Figure 4-4. The N1575Y and L1014F double mutations significantly reduced channel sensitivity
to permethrin and deltamethrin. (A) – (D) Tail currents induced by permethrin (1 µM) in
AaNav1-1, N1575Y, L1014F, and N1575+L1014F channels. (E) & (F) Percentage of channel
modification by permethrin and deltamethrin. Percentage of channels modified by pyrethroids
was calculated as described in Materials and Methods. The values of EC20 for permethrin are
0.12, 0.18, 1.0 and 9.8 µM for AaNav1-1, N1575Y, L1014F, and N1575Y+L1014F channels,
respectively. The values of EC20 for deltamethrin are 0.1, 0.11, 1.4, and 5.3 µM for AaNav1-1,
N1575Y, L1014F, and N1575Y+L1014F channels, respectively…………………………….109
Figure 4-5. The N1575Y and L1014S double mutations significantly reduced channel sensitivity
to permethrin and deltamethrin. (A) & (B) Percentage of channel modification by permethrin and
deltamethrin. Percentage of channels modified by pyrethroids was calculated as described in
Materials and Methods. The values of EC20 for permethrin are 0.12, 0.18, 0.8 and 7.0 µM for
AaNav1-1, N1575Y, L1014S, and N1575Y+L1014S channels, respectively. The values of EC20
for deltamethrin are 0.1, 0.11, 0.9, and 3.2 µM for AaNa v 1-1, N1575Y, L1014S, and
N1575Y+L1014S channels, respectively………………………………………………………111
xiv

Figure 4-6. Effects of the single V1016G mutation and the N1575Y+V1016G double mutations
on AaNav1-1 channel sensitivity to pyethroids. (A) Percentage modification by permethrin. (B)
Percentage modification by deltamethrin. Percentages of channel modification by permethrin (1
µM) or deltamethrin (1 µM) were determined using the method described in Materials and
Methods. *indicates a significant difference compared to AaNav1-1 using one-way ANOVA
with Scheffe’s post hoc analysis (p < 0.05)…………………………………………………...112
Figure 5-1. Localization of 20 A-to-I RNA editing sites in the Drosophila sodium channel. From
our collaborators in Zhejiang University in China and Yale University……………………….130
Figure 5-2. Differential sensitivities of DmNav sodium channel variants to deltamethrin. Sodium
channel variants were transiently expressed individually in Xenopus oocytes. Percentages of
channels modified by 1 µM pyrethroids was calculated using the equation
M={[Itail/(Eh−ENa)]/[INa/(Et−ENa)]}×100 (Tatebayashi and Narahashi, 1994),where Itail is the
maximal tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is
the reversal potential for sodium current determined from the current–voltage curve, INa is the
amplitude of the peak current during depolarization before pyrethroid exposure, and Et is the
potential of step depolarization. (Results from Dr. Zhaonong Hu)…………………………….131
Figure 5-3. Differential sensitivity of DmNav sodium channel variants to Av3. Sodium channel
variants were transiently expressed individually in Xenopus oocytes. Sodium channel sensitivity
to 250 nM of Av3 was measured by normalizing peak of non-inactivating current at end of 20 ms
d e p o l a r i z a t i o n t o p e a k o f t r a n s i e n t s o d i u m c u r r e n t . ( R e s u l t s f r om D r . R o n g
Gao)…………………………………………………………………………….……………….132
Figure 5-4. Topology of the sodium channel variant DmNa v 26. Four RNA editing sites
discovered in other DmNav sodium channels were shown in black and three non-RNA editing
amino acid changes were in gray.DmNav26 contains two optional exons, a and f, and two
mutually exclusive exons, d and k…………………………………………………………….133
Figure 5-5. Replacement of exon k with exon l significantly increased the sensitivity of
DmNav26 channel to deltamethrin. (A) Localization of the mutually exclusive exons k/l and the
alternative exon f. (B) Representative tail currents induced by 1 µM deltamethrin. (C) Percentage
of channel modification by deltamethrin. Sodium channel sensitivity to deltamethrin was tested
and analyzed as described in Materials and Methods. * Significant difference compared with
DmNav26 using one-way ANOVA with Scheffe’s post hoc analysis (p < 0.05)……………....134
Figure 5-6. Replacement of exon k with exon l increased the persistent sodium current. (A)
Voltage-dependence of activation. (B) Voltage-dependence of fast inactivation. (C)
+
Representative current traces of DmNav26 and exon l mutant channels induced by a step
depolarization from a holding potential of -120 mV to 0 mV. (D) Percentage of persistent current
xv

-

+

of DmNav26, exon f , and exon l channels. Percentage of persistent current was normalized to
the amplitude of peak sodium current. * Significant difference compared with DmNav26 using
one-way ANOVA with Scheffe’s post hoc analysis (p < 0.05)………………………………...136
Figure 5-7. Voltage-dependence of activation and fast inactivation of DmNav26 and Q299E,
F1363L, and S1977P mutants. (A) Position of all three non-RNA editing amino acid changes.
(B) Voltage-dependence of activation. (C) Voltage-dependence of fast inactivation. Voltagedependence of activation and fast inactivation were tested and analyzed as described in Materials
and Methods ……………......…………………………………………………………………..138
Figure 5-8. DmNav26 and Q299E, F1363L, and S1977P mutant channels have similar sensitivity
to deltamethrin. (A) Topological localization of all three none RNA editing amino acid changes.
(B) Tail current of DmNav26 induced by 1 µM deltamethrin and the formula of calculating
sodium channel percentage modification by pyrethroids. (C) Percentage modified channels of
DmNa v 26 and three none RNA editing mutant channels. Sodium channel sensitivity to
deltamethrin was tested and analyzed as described in Materials and Methods……………….140
Figure 5-9. Voltage-dependence of activation, fast inactivation, and slow inactivation of
DmNav26 and K437R, Q1296R, N1300D, and N1587S mutants. (A) Topological localization of
all four RNA editing events in DmNav26. (B) Voltage-dependence of activation. (C) Voltagedependence of fast inactivation. (D) Voltage-dependence of slow inactivation. Sodium channel
voltage-dependence of activation, fast inactivation and slow inactivation were tested and
analyzed as described in Materials and Methods……………………………………………...142
Figure 5-10. Q1296R substitution increased DmNa v 26 sensitivity to deltamethrin. (A)
Topological localization of the Q1296R mutation. (B) Representative tail currents of DmNav26
and Q1296R mutant channels induced by 1 µM deltamethrin. (C) Percentage modified channels
of DmNav26 and all four RNA editing event mutant channels. Sodium channel sensitivity to
deltamethrin was tested and analyzed as described in Materials and Methods. * Significant
difference compared with DmNav26 using one-way ANOVA with Scheffe’s post hoc analysis (p
< 0.05)……………………………………………………………………………...…………...144
Figure 5-11. Q1296R mutation increased DmNa v 26 sensitivity to Av3. (A) Topological
localization of Q1296R mutation. (B) Representative sodium current traces before and after Av3
application. Dashed line indicates the time point where sodium channel inactivation was
measured. (C) Percentage modification of DmNav26 and mutant channels by 250 nM Av3.
Percentage modification was calculated by normalizing the inactivation current at 20 ms point to
the peak sodium current. * Significant difference compared with DmNav26 using one-way
ANOVA with Scheffe’s post hoc analysis (p < 0.05)………………….……………………….146

xvi

CHAPTER 1
GENERAL INTRODUCTION

1

This study investigated the effects of auxiliary subunits, RNA editing and alternative
splicing of the Drosophila sodium channel on channel gating properties and sensitivity to
pyrethroid insecticides as well as the molecular mechanisms of pyrethroid resistance. Below I
give an introduction on the structure and function of mammalian and insect sodium channels and
also the mode of action of pyrethroid insecticides and insecticide resistance.
1.1 Sodium channels
1.1.1. History and structure of sodium channels
Hodgkin and Huxley were the pioneers who first recorded sodium currents from the
squid giant axon using voltage clamp techniques and demonstrated the three key features of
sodium currents: (1) voltage-dependent activation, (2) rapid inactivation, and (3) selective ion
conductance (Hodgkin and Huxley 1952). Functions of sodium channels have been extensively
characterized by the voltage clamp method without knowing the structure of sodium channels
throughout the 1960s and 1970s. Photoaffinity labeling and tetrodotoxin (TTX) binding
experiments proved that mammalian sodium channels are composed of a large polypeptide of
about 260 kDa and one or two small proteins of about 33-36 kDa (Agnew et al. 1980; Beneski
and Catterall 1980; Hartshorne and Catterall 1981; Hartshorne et al. 1982). The large protein is
the sodium channel α subunit, while the small proteins are sodium channel β subunits.
The sodium channel α subunit is a pore-forming subunit, which is composed of four
homologous domains (I–IV), each containing six hydrophobic transmembrane segments (S1–S6)
that are connected by both intra- and extra-cellular loops (Fig. 1-1) (Guy and Seetharamulu
1986; Noda et al. 1984). S5 and S6 segments and the reentrant loops between them form the pore
region, which has the reentrant loops as the narrow extracellular end and S6 as the intracellular
end. These four reentrant loops contain residues critical for ion selectivity (Catterall 2000).

2

Specifically, two rings formed by amino acids from the loops, EEDD and DEKA, are essential
for ion selectivity (Fig. 1-1) (Terlau et al. 1991). S1–S4 segments serve as the voltage-sensing
module. Sodium channels sense voltage changes of the cell membrane by the S4 segments,
which contain well arranged positively charged residues every third position. S4s move outward
or inward under the influence of the membrane electric field to initiate conformational changes
that open or close the pore (Catterall 1986; Guy and Seetharamulu 1986). The linker loop
connecting sodium channel domains III and IV has an IFMT particle that is important for sodium
channel fast inactivation (Fig. 1-1) (Rohl et al. 1999).
1.1.2. Sodium channel voltage-dependent gating properties
Voltage-gated sodium channels change conformations depending on membrane potential
change. In response to membrane depolarization, sodium channels open or activate, and sodium
ions flow into the cell. The S4 segment of each domain is critical for voltage sensing. Mutation
of the voltage sensor can profoundly alter the activation of sodium channels (Kontis et al. 1997).
S1-S4 are coupled with S6 via the S4-S5 linker helix, which enables channel opening and closing
upon changes in the membrane potential (Mannikko et al. 2002). Three comparable models have
been proposed to explain the coupling of voltage sensor with surrounding residues and the
movement of S4 during activation: the helical screw model, the transporter model, and the
paddle model (Borjesson and Elinder 2008). However, none of these models perfectly explain all
experimental results. Currently, the helical screw model is the one used to explain sodium
channel activation. This model proposes that the positive residues of S4 pair with negatively
charged residues in the neighboring segments (S1-S3). When the membrane is depolarized, this
pairing will be disrupted and the S4 segments will move outward, causing a conformational
change and sodium channel opening (Catterall and Epstein 1992).

3

After sodium channel activation, sodium channels become non-conducting or inactivate
within a few milliseconds. This fast inactivation is mediated by an inactivation particle formed
by residues in the linker connecting domains III and IV, which physically occludes the inner
pore, preventing ions from flowing into the cell (Catterall 2012). Sodium channel fast
inactivation is important for the repolarization of action potential (Goldin 2003).
Under prolonged depolarization, sodium channels enter into another inactivation state
called the slow inactivated state. Unlike fast inactivation, where recovery takes tens of
milliseconds, recovery from slow inactivation requires seconds to minutes of membrane
repolarization to return to a resting state (Goldin 2003; Vilin and Ruben 2001). Sodium channel
slow inactivation plays critical roles in controlling action potential patterns and spike frequency
adaptation in response to repetitive stimuli (Vilin and Ruben 2001). The molecular mechanism of
sodium channel slow inactivation is far from clear. Evidence has shown that amino acid residues
located in IVS4, IIS6, and the P-regions of all four domains are involved in sodium channel slow
inactivation (Mitrovic et al. 2000; Vilin and Ruben 2001).
1.1.3. Mammalian sodium channel α subunits
In mammals, the sodium channel is composed of one α subunit and one or two β
subunits. There are at least nine α subunit sodium channel genes, which encode distinct sodium
channel isoforms with different gating properties and expression patterns in various cell types,
tissues, and developmental stages (Catterall 2012; Goldin et al. 2000; Yu and Catterall 2003).
Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are mainly expressed in the central nervous system (CNS).
Nav1.4 is mainly expressed in skeletal muscle, while Nav1.5 is in cardiac muscle. Nav1.7,

4

Nav1.8, and Nav1.9 are mainly expressed in the peripheral nervous system (PNS) (Table 1-1)
(Goldin et al. 2000).
In addition to variable tissue distributions, Nav isoforms have also been shown to have
unique developmental profiles. Nav1.1, Nav1.2, and Nav1.6 are present at high levels in adult
CNS, but their presence in embryonic stages and neonatal stages is different. Nav1.1 becomes
detectable shortly after birth, whereas Nav1.2 becomes detectable during embryonic stages. Both
reach maximal levels during the adult stage. Nav1.6 is the most abundant isoform in adult CNS,
but the peak level is in late embryonic and early postnatal periods. Nav1.3 can only be detected
during embryonic and neonatal stages. Nav1.5 are detectable in neonatal skeletal muscles but are
replaced by Nav1.4 in adult skeletal muscles (Goldin 2001).
1.1.4 Mammalian sodium channel β subunits
In mammals, four β subunits (β1–β4) have been identified and functionally characterized
(Chahine et al. 2005). These subunits are transmembrane proteins with an extracellular
immunoglobulin (Ig) domain, a single transmembrane segment, and a small intracellular Cterminal domain (Fig. 1-2A). These β subunits not only serve as chaperone proteins, which
regulate trafficking and facilitate sodium channel folding on cell membrane, but also modulate
sodium channel gating (Isom 2002a). Additionally, these proteins play important roles in cell
aggregation and migration (Malhotra et al. 2000).
Different β subunits have differential expression patterns in vivo. For example, the β1
subunit is highly expressed in intermediate to large-diameter dorsal root ganglion (DRG) neurons

5

but less so in small diameter DRG neurons (Oh et al. 1995). The β2 subunit is detected at low
levels in all DRG neurons (Takahashi et al. 2003) but is abundant in the central nervous system
(CNS) (Gastaldi et al. 1998). Small-diameter DRG neurons are enriched with the β3 subunit
(Morgan et al. 2000). The β4 subunit is abundant in the CNS and in large-diameter DRG neurons
(Yu and Catterall 2003).
Each β subunit differentially modifies sodium channel gating properties in heterologous
expression systems. For example, β1 increased the Nav1.8 sodium current and caused negative
shifts in the voltage-dependence of activation and inactivation of both Nav1.7 and Nav1.8
channels (Vijayaragavan et al. 2004). Co-expression of β3 depolarized the voltage-dependence
of activation and inactivation (Cusdin et al. 2010). The β4 subunit induced negative shifts in the
+

voltage-dependence of activation of several Na channels, including Nav1.1, Nav1.2, Nav1.4,
and Nav1.6 channels (Aman et al. 2009; Chen et al. 2008b; Yu et al. 2003). Functional
modification of Nav1.6 and Nav1.8 channels by β1-4 subunits has also been extensively
investigated in HEK293 cells (Zhao et al. 2011). The β1 subunit increased Nav1.8 peak current
by about 2.3-fold, while β2 and β4 did not alter the peak current of Nav1.8 channels. The β3
subunit decreased Nav1.8 peak current about 31%, while β1-4 did not alter the peak current of
Nav1.6 channels. For gating modifications, the β1 subunit caused negative shifts in the voltagedependence of activation and inactivation of Nav1.8 channels but not Nav1.6 channels. However,
the β4 subunit caused significant negative shifts in the voltage-dependence of activation and

6

inactivation of both Nav1.8 and Nav1.6 channels. There was no evidence that β2 and β3 modify
gating properties of either Nav1.8 or Nav1.6 (Zhao et al. 2011).
1.1.5. Insect sodium channels
In contrast to the multiple sodium channel genes in mammals, insects have only one
sodium channel gene that encodes the α subunit equivalent of mammalian sodium channels
(Dong 2010; Loughney et al. 1989). For example, para (DmNav) is the only confirmed sodium
channel gene in D. melanogaster (Feng et al. 1995). Most insect species have only one DmNavlike gene, such as the Vssc1 gene in houseflies (Ingles et al. 1996) and the BgNav gene in
cockroaches (Dong and Scott 1994). However, functionally distinct sodium currents have been
documented in insect neurons, indicating the existence of sodium channels with different gating
properties (Byerly and Leung 1988; Defaix and Lapied 2005; Grolleau and Lapied 2000; Lapied
et al. 1999; Le Corronc et al. 1999; O'Dowd 1995; Saito and Wu 1993; Schafer et al. 1994;
Wicher et al. 2001). How do insects generate functionally distinct sodium channels with only one
gene? Extensive alternative splicing has been shown to generate diverse sodium channel
transcripts in many insect species (Fletcher et al. 2011; He et al. 2012; Lin et al. 2009; Liu et al.
2001; Olson et al. 2008; Tan et al. 2002b; Zhang et al. 2011). On the other hand, RNA editing
events regulating sodium channel gating properties are mainly reported for the transcripts of
DmNav and BgNav (Liu et al. 2004; Olson et al. 2008; Song et al. 2004).
1.1.6. Insect sodium channel auxiliary subunits
Insects lack any orthologs of mammalian sodium channel β subunits (Littleton and
Ganetzky 2000). TipE is critical for sodium channel expression and neuronal activities and

7

functions as an auxiliary subunit for insect sodium channels (Feng et al. 1995; Warmke et al.
3

-

1997). [H ]saxitoxin binding studies of insect embryonic neurons indicated that tipE mutant
flies have reduced sodium channel density (Jackson et al. 1986). Consistently, whole-cell patch
recording indicated that the sodium current decreased about 40% to 60% in dissociated
-

embryonic neurons of tipE mutant flies (O'Dowd and Aldrich 1988). TipE also increased the
peak current of sodium channels transiently expressed in Xenopus oocytes (Derst et al. 2006;
Feng et al. 1995; Warmke et al. 1997). In addition to TipE, there are four TipE-homologous
genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species (Derst et
al. 2006). TEH1 is exclusively expressed in the nervous system, while transcripts of TipE and
TEH2-4 are detected in both neuronal and non-neuronal tissues (Derst et al. 2006). All four TEH
proteins differentially modified one D. melanogaster sodium channel variant expressed in
Xenopus oocytes (Derst et al. 2006).
Structurally different from mammalian sodium channel β subunits, TipE and TEH1-4
have two transmembrane domains, one extracellular loop, and intracellular C- and N-terminus
are (Fig. 1-2B). The sequence of the extracellular loops of TEH3 and TEH4 are completely
different from those of TipE and TEH1-2. There are eleven conserved cysteine residues in the
extension extracellular loops of TEH3 and TEH4. This feature is similar to the typical
extracellular epidermal growth factor (EGF) domain, which contains six conserved cysteine
residues involved in three disulfide bonds.
While the functional importance of mammalian sodium channel β subunits has been
extensively examined, the role of TEH1-4 in modulating gating and pharmacological properties
of insect sodium channel variants is largely unknown. Functional examination of TEH1-4 in
Xenopus oocytes with a DmNav sodium channel variant showed that TEH1-3, but not TEH4,
8

enhanced the amplitude of sodium channel current in Xenopus oocytes (Derst et al. 2006). TEH1
induced a negative shift of voltage-dependent fast inactivation and slowed the rate of recovery
from fast inactivation (Derst et al. 2006). However, the effects of TEH1 on activation, and the
gating modifications by TEH2-4, were not examined. Whether TEH1-4 proteins modify sodium
channel variants in a variant-specific manner is not known.
1.2 Alternative splicing and RNA editing
1.2.1. Alternative splicing
Alternative splicing is a post-transcriptional process during which the exons of the RNA
are selectively used. Different usage of alternative exons will produce differential variants. Until
now, eleven alternative splice exons in total have been identified in DmNav transcripts: a, b, c/d,
i, j, e, f, h, l/k (Lee et al. 2002; O'Dowd 1995; Thackeray and Ganetzky 1994; Thackeray and
Ganetzky 1995). Seven of them (a, b, i, j, e, f, h) are optional in the intracellular linkers, while
the other two pairs, c/d and l/k, are mutually exclusive in the transmembrane segments (Fig. 1-3).
Those alternative splicing exons are mostly conserved in different insect species, suggesting
important roles in modulating sodium channel function (Dong 2007; Dong 2010). However,
alternative exon usage varies in different insect species. For example, more than 60% of DmNav
and Vssc1 transcripts contain exon b (Lee et al. 2002), but less than 20% of BgNav transcripts
contain exon b (Song et al. 2004).
The functional importance of alternative splicing exons in modulating sodium channel
gating properties can be investigated in Xenopus oocytes using the two-electrode voltage clamp
technique. It has been reported that inclusion of optional exons modulates the voltage
dependence of activation and/or inactivation of DmNav channels isolated from late embryonic
9

stage of D. melanogaster (Lin et al. 2009). For example, the inclusion of exon f caused a
hyperpolarizing shift in the voltage-dependence of activation, whereas the inclusion of exon j or
exon e induced a depolarizing shift of activation. Sodium channel variants containing exon h
normally had more depolarized voltage-dependence of inactivation, while exon j containing
variants had more negative voltage dependence of inactivation (Lin et al. 2009). Alternative
+

splicing is also involved in the generation of persistent Na currents, which are thought to be
involved in the boosting of excitatory synaptic inputs, acceleration of firing rates, and promotion
of oscillatory neuronal activates (Li and Bennett 2003; Li et al. 2004). Besides, inclusion of exon
b in BgNav sodium channel variants significantly decreased sodium channel expression in
Xenopus oocytes (Song et al. 2004).
Alternative splicing can generate pharmacologically distinct sodium channels. Two
mutually exclusive exons G1/G2 located in ІІІS3-S4 of cockroach sodium channel variants
correspond to l/k in DmNav. Exon G2 is found in BgNav2-1, whereas BgNav1-1 has exon G1.
BgNav2-1 was about 100-fold less sensitive to deltamethrin, than BgNav1-1. Exons G2/G1 were
partially responsible for the differential pyrethroid sensitivity (Tan et al. 2002b). Deletion of
G1111 in BgNav2-1, as a result of alternative splicing, reduced channel sensitivity to pyrethroids
(Du et al. 2009).
1.2.2. RNA editing
RNA editing is a post-transcriptional process during which one nucleotide base is altered
or converted to another nucleotide, or a nucleotide is deleted or inserted. Both A-to-I and U-to-C
editing events have been reported for insect sodium channel genes (Liu et al. 2004; Olson et al.
2008; Song et al. 2004). Initially, ten A-to-I editing sites were identified in DmNav transcripts by
10

sequence analysis and enzyme digestion of partial DmNav sodium channel cDNAs (Hanrahan et
al. 2000; Palladino et al. 2000; Reenan et al. 2000). Since then, more A-to-I editing sites have
been discovered in DmNav transcripts (Olson et al. 2008). In the cockroach sodium channel
BgNav, two A-to-I and three U-to-C RNA editing events were identified in the analysis of fulllength cDNA clones (Liu et al. 2004; Song et al. 2004).
Recently, 20 A-to-I RNA editing sites were identified in sodium channel transcripts from
different body parts of the adult D. melanogaster by high throughput sequencing (Fig. 1-4, from
Dong lab’s collaborators in Yale University and Zhejiang University in China). However, the
functional consequences of these RNA editing events have not been examined.
Although the role of RNA editing has not been studied in vivo, electrophysiological
studies in Xenopus oocytes provide valuable information about functional modifications due to
RNA editing events. For example, one U-to-I editing event in BgNav1-1 generated one amino
acid change, P1285L, that was responsible for the negative shifts in voltage-dependence of
activation and inactivation, while another A-to-I event in BgNav2-1 induced a negative shift in
the voltage-dependence of activation (Song et al. 2004). A large persistent sodium current is
detected because of an U-to-C RNA editing event at the C-terminal domain of both BgNav and
DmNav channels (Liu et al. 2004). An A-to-I RNA editing event was responsible for a negative
shift in the voltage-dependence of activation of the DmNav5-1 channel (Olson et al. 2008).
1.3. Pyrethroids
1.3.1. Pyrethrum and pyrethroids

11

Pyrethrins are natural compounds found in pyrethrum extracted from flowers of
Chrysanthemum species. Pyrethrins were found with insecticidal activity (Casida 1980).
However, due to their instability in the sun, pyrethrum and pyrethrins are mainly used to control
household insect pests such as mosquitoes and houseflies.
Pyrethroid insecticides are a large class of synthetic insecticides derived structurally from
the natural pyrethrins. The first several pyrethroids were synthesized from 1940 to 1970 (Elliott
1980). A few of them, such as allethrin, tetramethrin, and resmethrin, exhibited excellent
insecticidal activity. Permethrin was synthesized and confirmed potent and photostable in 1973
(Elliott et al. 1973). A few more pyrethroids, like deltamethrin and cypemethrin, were
synthesized in the following years (Hall 1978). Pyrethroid insecticides are widely used in
controlling agriculturally and medically important pests worldwide.
1.3.2. Type I and type II pyrethroids
Pyrethroids can be divided into two types (Type І and type ІІ) based on their distinct
poisoning symptoms, effects on nerve preparation and chemical structures (Gammon et al. 1981;
Lawrence and Casida 1982). Those pyrethroids that do not have a cyano group at the
phenxylbenzyl alcohol position are designated as type І pyrethroids; examples include allethrin,
tetramethrin, bioresmethrin, and permethrin. Pyrethroids that have an α cyano group are called
type ІІ pyrethroids; examples include deltamethrin, cyphenothrin, cypermethrin, and fenvalerate
(Fig. 1-5).
At the animal level, type І pyrethroids induce hyperexcitation, ataxia, convulsions and
eventually paralysis, while type ІІ pyrethroids cause tremors and paralysis. Electrophysiology
studies on nerve preparation showed that type І pyrethroids induce repetitive discharges in

12

response to a single stimulus, while type ІІ pyrethroids cause a membrane depolarization
followed by suppression of action potentials (Narahashi 1986).
1.3.3. The mode of action of pyrethroids
Pyrethroids bind, predominantly, to open-state sodium channels and inhibit sodium
channel inactivation and deactivation, which result in prolonged opening of sodium channels
(Dong 2007; Narahashi 1987; Soderlund 2012; Soderlund and Bloomquist 1989; Vijverberg and
van den Bercken 1990). In voltage-clamp experiments, pyrethroids induce a slowly decaying
sodium current – tail current, upon repolarization. The kinetics of tail current decay are different
for type I and type II pyrethroids: the decay of tail currents induced by type II pyrethroids is
slower than that induced by type I pyrethroids, which may account for their different actions on
the nervous system (described above) (Dong 2007; Narahashi 1986). Application of pyrethroids
prolonged the open state and altered channel activation kinetics (Bloomquist 1993; Narahashi
1992). One interesting feature of pyrethroids is that, pyrethroids are much more potent at low
temperatures than at high temperatures (Ginsburg and Narahashi 1999; Motomura and Narahashi
2000; Song and Narahashi 1996; Unkiewicz-Winiarczyk and Gromysz-Kalkowska 2012).
1.3.4. Knockdown resistance (kdr)
Pyrethroid insecticides are widely used to control both agriculturally and medically
important insects because of their high insecticidal activity and low mammalian toxicity
(Narahashi 2000). However, intensive use of pyrethroid insecticides leads to the development of
resistance. One of the important mechanisms of pyrethroid resistance is called knockdown
resistance (kdr), which has been documented globally in almost all major arthropod pests and
disease vectors (Rinkevich et al. 2013; Soderlund and Bloomquist 1990). It is caused by sodium
channel mutations which reduce or abolish the sensitivity of insect sodium channels to

13

pyrethroids (Dong 1997; Dong 2007; Soderlund and Bloomquist 1990). So far, more than 40 kdr
mutations have been documented in various arthropod species (Rinkevich et al. 2013). Some kdr
mutations have been detected only in certain insect species, while others were found in multiple
insect species (Rinkevich et al. 2013). The most common kdr mutation is a leucine (L) to
phenylalanine (F), histidine (H), or serine (S) change in the segment 6 of domain ІІ (ІІS6).
Pharmacological studies in Xenopus oocytes showed that this L to F/H mutation in ІІS6
significantly reduces DmNav, Musca domestica sodium channel MdNav and Blettalla germanica
sodium channel BgNav channels sensitivity to pyrethroids (Liu et al. 2002; Smith et al. 1997;
Vais et al. 2000; Zhao et al. 2000). Sodium channel kdr mutations have been used as molecular
markers for monitoring of kdr resistance in field populations (Brun-Barale et al. 2005; Drali et al.
2012; Henry-Halldin et al. 2012; Kim et al. 2005; Martinez-Torres et al. 1998).
1.3.5. The pyrethroid receptor sites
Electrophysiological and pharmacological studies suggested that pyrethroids have a
distinct receptor site on the sodium channel (Bloomquist and Soderlund 1988; Brown et al. 1988;
Lombet et al. 1988; Takeda and Narahashi 1988). It is technically difficult to characterize the
pyrethroids receptor site(s) on sodium channels in a ligand-receptor binding assay using insect
nerve tissues because of the high lipophilicity of pyrethroids, which causes extremely high
nonspecific binding to membranes (Dong and Scott 1994; Pauron et al. 1989; Rossignol 1988).
With the accumulated knowledge in the molecular basis of sodium channel interaction
with pyrethroids as well as the availability of crystal structures of a potassium channel and a
bacterial sodium channel, two pyrethroid receptor sites have been proposed (Du et al. 2013;
O'Reilly et al. 2006). The first pyrethroid receptor was proposed in the open state of the house fly
sodium channel based on the crystal structure of voltage-gated potassium channel (O'Reilly et al.
14

2006). This model indicated that pyrethroids bind to a triangle pocket composed of the linker
connecting S4 and S5 in domain II, domain II S5, and domain III S6. The second pyrethroid
receptor was built on a mosquito sodium channel based on the crystal structures of a voltagegated potassium channel and a bacterial sodium channel (Du et al. 2013). This second proposed
pyrethroid receptor site is composed of the linker connecting S4 and S5 in domain I, domain I
S5, and domain II S6 (Du et al. 2013).
More than 20 kdr mutations are located in one of the two pyrethroid-receptor sites (i.e.,
Site 1 and Site 2) and confer resistance by reducing pyrethroid binding. However, many other
kdr mutations are not located close to these receptor sites, suggesting that they may confer
pyrethroid resistance by receptor site-independent mechanisms, or they are located in a new
pyrethroid receptor site that has yet not been identified.
1.4. Differential sensitivity of sodium channel variants to pyrethroids and Av3
Av3 is a short polypeptide toxin from the venom of the sea anemone Anemonia. viridis
(Moran et al. 2007). Av3 targets on sodium channels and inhibits channel inactivation (Moran et
al. 2007). Sodium channel variants were isolated from adult D. melanogaster and they exhibited
diverse gating properties in Xenopus oocytes (Olson et al. 2008). Pharmacological
characterization of these DmNav variants in Xenopus oocytes showed that these DmNav variants
had different sensitivities to both Av3 and a pyrethroid insecticide, deltamethrin (Fig. 1-7 and
Fig. 1-8, results from Drs. Zhaonong Hu and Rong Gao). Differential sensitivities of sodium
channel variants to neurotoxins provide valuable information for understanding the molecular
basis of neurotoxin and sodium channel interaction.

15

Table 1-1. Tissue distribution of nine mammalian voltage-gated sodium channel α subunits (Goldin et al.
2000; Yu and Catterall 2003).

Isoform

Species

Nav1.2

Rat
Guinea pig
Rat
Human

Nav1.3

Rat

Nav1.1

Nav1.4
Nav1.5
Nav1.6

Nav1.7
Nav1.8
Nav1.9

Primary Tissue Distribution
CNS, PNS
CNS
CNS

Rat
Human
Rat
Human
Rat
Human
Mouse
Guinea pig
Rat
Human
Rabbit
Mouse
Dog
Rat
Mouse
Human

16

Skeletal muscle
Skeletal and heart muscle

CNS, PNS

PNS
PNS
PNS

Figure 1-1. Topology of the voltage-gated sodium channel α subunit. The α subunit is composed
of four homologous domains (I-IV), each of which contains six transmembrane segments (S1S6). Positively charged amino acid residues in S4s are represented as +, which serve as voltage
sensors. The S5 and S6 segments and the P-region between them together form the inner pore of
the sodium channel. Amino acid residues EEDD and DEKA in the linker connecting S5 and S6
in each domain form two rings that are essential for ion selectivity. The black dot in the linker
connecting domains III and IV represents the fast inactivation particle, IFMT (isoleucine,
phenylalanine, methionine, threonine), which is thought to occlude the pore. (Modified from
Catterall, 2000)

17

Figure 1-2. Structure of mammalian sodium channel β subunits and insect sodium channel
auxiliary subunits. (A) Structure of β subunits. Mammalian sodium channel β subunits are small
transmembrane proteins that possess an extracellular immunoglogulin (Ig) domain, a single
transmembrane segment, and a short intracellular C-terminal domain. (B) Structure of
TipE/TEH1-4. Insect sodium channel auxiliary subunit has two transmembrane segments
connected by one extracellular linker and both N- and C-terminus are intracellular.

18

Figure 1-3. Distribution of alternative exons on DmNav transcripts. A schematic diagram of the
DmNav sodium channel labeled with names and locations of 11 alternative exons indentified in
DmNav transcripts. Exons a, b, e, f, h, I, and j are optional exons. Exons d/c and l/k are two pairs
of mutually exclusive exons. (Modified from Olson et al., 2008)

19

Figure 1-4. Localization of 20 A-to-I RNA editing sites in the Drosophila sodium channel. From
our collaborators in Zhejiang University in China and Yale University.

20

Figure 1-5. Chemical structure of type I and type II pyrethroids. Cyano group in type II
pyrethroids is labeled as *.

21

Figure 1-5. (Cont’d)

22

Figure 1-6. Differential sensitivity of DmNav sodium channel variants to Av3. Sodium channel
variants were transiently expressed individually in Xenopus oocytes. Sodium channel sensitivity
to 250 nM of Av3 was measured by normalizing peak of non-inactivating current at end of 20 ms
depolarization to peak of transient sodium current. (Results from Dr. Rong Gao).

23

Figure 1-7. Differential sensitivity of DmNav sodium channel variants to deltamethrin. Sodium
channel variants were transiently expressed individually in Xenopus oocytes. Percentages of
channels modified by 1 µM pyrethroids was calculated using the equation
M={[Itail/(Eh−ENa)]/[INa/(Et−ENa)]}×100 (Tatebayashi and Narahashi, 1994),where Itail is the
maximal tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is
the reversal potential for sodium current determined from the current–voltage curve, INa is the
amplitude of the peak current during depolarization before pyrethroid exposure, and Et is the
potential of step depolarization. (Results from Dr. Zhaonong Hu).

24

CHAPTER 2
DIFFERENTIAL EFFECTS OF TIPE AND A TIPE-HOMOLOGOUS PROTEIN ON
MODULATION OF GATING PROPERTIES OF SODIUM CHANNELS FROM
DROSOPHILA MELANOGASTER

25

2.1. Abstract
β subunits of mammalian sodium channels play important roles in modulating the
expression and gating of mammalian sodium channels. However, there are no orthologs of β
subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its
orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there
are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in
other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various
insect sodium channels expressed in Xenopus oocytes. However, limited information is available
on how these proteins modulate the gating of sodium channels, particularly sodium channel
variants generated by alternative splicing and RNA editing of a single gene. In this study, I
compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel
splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1
enhanced the amplitude of sodium current and accelerated current decay of all three sodium
channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage dependence of
activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not
alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast
inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that
TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the
recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow
inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of
insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating
sodium channel function and neuronal excitability in vivo.

26

2.2. Introduction
Voltage-gated sodium channels are transmembrane proteins that are critical for the
initiation and propagation of action potentials in neurons and other excitable cells (Catterall
2000). Upon membrane depolarization, sodium channels open, resulting in sodium ion influx and
further depolarization of the membrane potential. This process is called channel activation,
which is responsible for the rapidly rising phase of action potential. After channel opening,
sodium channels inactivate rapidly, within a few milliseconds, in a process known as fast
inactivation. Fast inactivation plays an important role in the termination of action potentials.
Furthermore, in response to prolonged depolarization (seconds to minutes), sodium channels
progressively enter into more stable, slow-inactivated states. This process is known as slow
inactivation, which is important for regulating membrane excitability, action potential patterns
and spike frequency adaptation (Vilin and Ruben 2001).
Mammalian sodium channels are composed of a pore-forming α subunit and one or more
β subunits. Sodium channel α subunits have four homologous domains (I–IV), each containing
six transmembrane segments (S1–S6). Mammals have nine α-subunit genes which encode
sodium channel isoforms with different gating properties and different expression patterns in
various cell types, tissues, and developmental stages, presumably to fulfill unique physiological
functions in specific neuronal and non-neuronal cells (Catterall 2000; Goldin et al. 2000; Yu and
Catterall 2003). Four homologous β subunits (β1–β4) have been identified and characterized
(Chahine et al. 2005). They are small transmembrane proteins that possess an extracellular
immunoglobulin (Ig) domain, a single transmembrane segment, and a short intracellular Cterminal domain. β subunits are known to modulate both sodium channel gating and expression
in in vitro expression systems (Isom 2002b). A particular β subunit can have variable effects on

27

different sodium channel isoforms. For instance, β2 causes a depolarizing shift in the steady-state
inactivation of Nav1.2 channels, but has little effect on Nav1.3 channels (Meadows et al. 2002;
Qu et al. 2001). Different β subunits can also have different effects on a given sodium channel
isoform. For instance, β1, β2 and β3 all accelerated fast inactivation kinetics of Nav1.8 channels;
β1 enhanced peak sodium current of Nav1.8 channels and causes hyperpolarizing shifts in the
voltage-dependences of activation and inactivation, whereas β2 and β3 had no effect on peak
sodium current and instead caused depolarizing shifts in the voltage-dependence of activation
and inactivation of Nav1.8 channels (Vijayaragavan et al. 2004).
In contrast to mammals, insects appear to have only a single sodium channel gene that
encodes the α-subunit equivalent of mammalian sodium channels (Dong 2010; Loughney et al.
1989). Despite having only a single gene, insects employ alternative splicing and RNA editing to
generate many sodium channel variants with different gating and pharmacological properties
(Dong 2007; Dong 2010). Interestingly, there are no orthologs of mammalian β subunit in insects
(Littleton and Ganetzky 2000). Instead, a transmembrane protein, TipE, is considered to be an
auxiliary subunit of insect sodium channels because it increases the functional expression of
-

insect sodium channels in Xenopus oocytes; and TipE mutants exhibit a temperature-sensitive
paralytic phenotype, similar to sodium channel mutants (Dong 2010; Feng et al. 1995; Suzuki et
al. 1971; Warmke et al. 1997).
Derst and associates identified four TipE-homologous genes (TEH1-4) in the genome of
Drosophila melanogaster (Derst et al. 2006). TEH1 is expressed in the central nervous system,
whereas the transcripts of the other three were also detected in non-neuronal tissues, such as fat
body and gut (Derst et al. 2006). TEH1-3 proteins have been shown to increase the amplitude of
28

sodium currents of a Drosophila sodium channel in Xenopus oocytes (Derst et al. 2006). TEH1
has also been shown to shift the voltage-dependence of fast inactivation in the hyperpolarizing
direction and slow the recovery from fast inactivation of a Drosophila sodium channel (different
from sodium channel variants in this study) (Derst et al. 2006). TipE accelerates the inactivation
kinetics of the same Drosophila sodium channel (Warmke et al. 1997). However, the extent of
TipE- or TEH1-mediated gating modification and whether their effects are variant-specific
remains unclear.
Sodium channels are the primary target of pyrethroid insecticides (Narahashi 2000;
Soderlund and Bloomquist 1989). Because of the involvement of sodium channel mutations in
pyrethroid resistance, intense research has been carried out in the past two decades to
functionally express and characterize the effects of pyrethroids on the gating properties of insect
sodium channels in Xenopus oocytes (Dong 2010; Soderlund 2005). Prior to this study, almost
all functional and pharmacological analyses of insect sodium channels were conducted by coexpression of insect sodium channels with TipE, and it is not clear whether TipE or TEH1
modulate the action of pyrethroids.
In a previous study, 33 functional Drosophila sodium channel (DmNav) splice variants
were identified with a wide range of voltage dependences of activation and inactivation
(O'Donnell-Olson et al. 2008). In this study, we used three of these splice variants, DmNav9-1,
DmNav22 and DmNav26, to compare the effects of TipE and TEH1 on DmNav channels. We
chose these three variants because, in the absence of TipE or TEH1, they generate sufficient
currents for electrophysiological analysis, which made it possible to evaluate the gatingmodifying effects of TipE or TEH1. In addition, these variants belong to three different splice

29

types and exhibit different functional properties (O'Donnell-Olson et al. 2008), which potentially
allows us to determine variant-specific gating modulation by TipE and/or TEH1. Our results
show that, like TipE, TEH1 enhanced the expression of sodium currents and accelerated current
decay of all three variants. Furthermore, we found that TEH1 extensively modified sodium
channel functional properties of all three variants, whereas TipE only modified the gating of one
of the variants. TEH1, but not TipE, also reduced DmNav9-1 sensitivity to deltamethrin by
reducing the duration of sodium channels in the open state. Our findings raise the possibility that
TEH1 may play a broader role in regulating sodium channel gating and neuronal excitability in
vivo.
2.3. Materials and Methods
2.3.1. Xenopus oocyte expression system
Oocytes were obtained surgically from female Xenopus laevis (Nasco, Ft. Atkinson. WI) and
incubated with 1 mg/ml Type IA collagenase (Sigma Co., St. Louis, MO) in Ca

2+

-free ND-96

medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5). Follicle still
remaining on the oocytes following digestion was removed with forceps. Isolated oocytes were
incubated in ND-96 medium containing 1.8 mM CaCl2 supplemented with 50 µg/ml gentamicin,
5 mM pyruvate, and 0.5 mM theophylline (Goldin 1992). Healthy stage V-VІ oocytes were used
for cRNA injection. TipE/TEH1 cRNA or H2O (as control) was injected together with DmNav
cRNA at a 1:1 ratio.
2.3.2. Electrophysiological recording and analysis
Methods for two-electrode recording and data analysis were similar to those described
previously (Tan et al. 2005). The borosilicate glass electrodes were filled with filtered 3 M KCl
30

in 0.5% agarose and had a resistance of 0.5 to 1.0 MΩ. The recording solution was ND-96
recording solution (96 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, and 10 mM
HEPES, pH adjusted to 7.5 with NaOH). Sodium currents were measured with a Warner
OC725C oocyte clamp amplifier (Warner Instrument, Hamden, CT) and processed with a
Digidata 1440 (Axon Instruments Inc., Foster City, CA). Data were sampled at 50 kHz and
filtered at 2 kHz. Leak currents were corrected by p/4 subtraction. pClamp 10.2 software (Axon
Instruments Inc., CA) and Origin 8.0 software were used for data acquisition and analysis.
The voltage dependence of sodium channel conductance (G) was calculated by
measuring the peak current at test potentials ranging from mV to +65 mV in 5
−80

mV

increments and divided by (V − V rev), where V is the test potential and Vrev is the reversal
potential for sodium current. Peak conductance values were normalized to the maximal peak
conductance (Gmax) and fitted with a two-state Boltzmann equation of the form G/Gmax = [1 +
−1

exp (V − V1/2)/k] , in which V is the potential of the voltage pulse, V1/2 is the voltage for halfmaximal activation, and k is the slope factor.
The voltage dependence of sodium channel fast inactivation was determined by using 100
ms inactivating pre-pulses ranging from -120 mV to 0 mV in 5 mV increments from a holding
potential of −120 mV, followed by test pulses to -10 mV for 20 ms. The peak current amplitude
during the test pulse was normalized to the maximum current amplitude and plotted as a function
of the pre-pulse potential. Data were fitted with a two-state Boltzmann equation of the form
−1

I/Imax = [1 + (exp(V − V1/2)/k)] , in which I is the peak sodium current, Imax is the maximal

31

current evoked, V is the potential of the voltage pre-pulse, V1/2 is the half-maximal voltage for
inactivation, and k is the slope.
The voltage dependence of sodium channel slow inactivation was measured with 60 s
conditioning pulses ranging from −100 mV to 0 mV in 10 mV increments, followed by
repolarization to a holding potential of -120 mV for 100 ms to remove fast inactivation, and at
last a test pulse of -10 mV for 20 ms. The peak current amplitude during the test depolarization
was normalized to the amplitude of maximum current during test pulse and plotted against the
conditioning potential. Data were fitted with a two-state Boltzmann equation as above for fast
inactivation.
Development of fast inactivation was measured by holding oocytes at -120 mV, followed
by a pre-pulse depolarization to -45 mV for 0 to 80 ms, and then a test pulse of -10 mV for 20 ms
to measure the fraction of sodium current inactivated during the pre-pulse. The peak current
during the test pulse was divided by the peak current which has a pre-pulse duration of 0 ms and
plotted as a function of duration time of pre-pulse. Time constant (τ) was calculated by fitting the
plot with a single exponential decay function.
Recovery from fast inactivation was tested with a conditioning depolarization of -10 mV
for 100 ms, which will drive all sodium channels into the fast inactivated state, then
repolarization to -70 mV for 0 - 20 ms followed by a 20-ms test pulse to -10 mV. The peak
current during the test pulse was divided by the peak current during the conditioning pulse and
plotted as a function of duration time between two pulses. Time constant (τ) of recovery from
fast inactivation was calculated by fitting the plot with a single exponential function.
Development of slow inactivation was measured by holding oocytes at -120 mV,
followed with a -10 mV pre-pulse depolarization for 0 to 25 s, then repolarization to -120 mV for
32

100 ms to remove fast inactivation, and a test pulse of -10 mV for 20 ms to measure the fraction
of sodium current inactivated during the pre-pulse. The peak current during the test pulse was
divided by the peak current which has a pre-pulse duration of 0 ms and plotted as a function of
duration of pre-pulse. Time constant (τ) was calculated by fitting the plot with an exponential
decay function.
Recovery from slow-inactivation was measured by holding oocytes at -120 mV, followed
by a pre-pulse to -10 mV for 60 s to drive sodium channels into the slow inactivated state,
followed by repolarization to -120 mV for 0 to 30 s, and finally a test pulse to -10 mV for 20 ms.
The peak current during the test pulse was divided by the peak current which has a repolarizing
duration of 30 s and plotted as a function of duration between the pre and test pulses. Recovery
from slow inactivation was well fitted by a double exponential function.
2.3.3. Measurement of sodium channel sensitivity to deltamethrin
The method for application of deltamethrin in the recording system was identical to that
described by Tan et al. (Tan et al. 2002a). The effect of deltamethrin was measured 10 min after
toxin application. Deltamethrin-induced tail currents were recorded with a 100-pulse train of 5
ms step depolarizations from -120 to 0 mV at 66.7 Hz (Vais et al. 2000). Additionally,
deltamethrin-induced tail currents were measured using a single pulse protocol with a 500-ms
step depolarization from -120 mV to -10 mV. The percentage of channels modified by
deltamethrin was calculated using the equation M = {[Itail/(Eh − ENa)]/[INa/(Et − ENa)]}×100
(Tatebayashi and Narahashi 1994), where Itail is the maximal tail current amplitude, Eh is the
potential to which the membrane is repolarized, ENa is the reversal potential for sodium current

33

determined from the current-voltage curve, INa is the amplitude of the peak current during
depolarization before pyrethroids exposure, and Et is the potential of step depolarization.
2.3.4. Chemicals
Deltamethrin was kindly provided by Bhupinder Khambay (Rothamsted Research,
Harpenden, UK). Deltamethrin was dissolved in dimethyl sulfoxide (DMSO). The working
concentration was prepared in ND-96 recording solution immediately prior to experiments. The
concentration of DMSO in the final solution was <0.5%, which had no effect on the function of
sodium channels.
2.3.5. Statistical analysis
Data were analyzed using Origin 8.0 software. Results are reported as mean ± SEM. Data
were analyzed using one-way analysis of variance (ANOVA), if ANOVA shows a significant
effect, Scheffe’s post hoc comparisons were used to compare the means. Results were considered
to be significant when p < 0.05.

2.4. Results
2.4.1. TipE and TEH1 increased the peak sodium current and accelerated the current decay of all
three DmNav variants
To compare the expression of sodium current in Xenopus oocytes, equal amounts of
cRNA synthesized in vitro from the DmNav9-1, DmNav22, or DmNav26 plasmids were injected
into oocytes with or without TipE or TEH1 cRNA. Sodium current was recorded 48 hours after
injection by step depolarizations to a series of voltages ranging from -80 mV to +25 mV in 5-mV

34

increments with a holding potential of -120 mV. Both TipE and TEH1 increased the amplitude of
maximum peak sodium current of all three variants by 4 to 9 fold (Fig. 2-1A and B).
We then measured the effect of TipE and TEH1 on the decay of sodium currents for all
three variants. Current decay was well fitted by a single exponential equation with or without
TipE or TEH1. TipE significantly increased the rate of current decay of all three variants
between -40 mV and -5 mV (Fig. 2-2A, C, and E). Similarly, TEH1 also slightly accelerated
current decay for all three variants, but over different voltage ranges (Fig. 2-2B, D, and F). TEH1
affected the current decay of DmNav22 channels over a broader voltage range (between -40 mV
to 10 mV) than the other two variants which were affected between -40 mV and -30 mV for
DmNav9-1, and -40 mV and -20 mV for DmNav26 (Fig. 2-2B, D, and F).
2.4.2. Differential effects of TEH1 and TipE on the voltage-dependence of activation and fast
inactivation
Co-expression of TEH1with DmNav9-1 induced a 12 mV hyperpolarizing shift in the
voltage-dependence of activation (Fig. 2-3A and Table 2-1). Similarly, significant
hyperpolarizing shifts were also detected with co-expression of TEH1 with DmNav26 or
DmNav22 (Table 2-1). However, co-expression of TipE did not modify the voltage-dependence
of activation of any variants (Fig. 2-3A and Table 2-1).
Co-expression of TEH1 with DmNav9-1 induced a 9 mV hyperpolarizing shift in the
voltage-dependence of fast inactivation compared with DmNav9-1 alone (Fig. 2-3B and Table 21). Similarly, co-expressing TEH1 with DmNav26 or DmNav22 also significantly shifted the
voltage-dependence of fast inactivation in the hyperpolarizing direction (Table 2-1). TipE did not
35

alter the voltage-dependence of fast inactivation of DmNav9-1 or DmNav22 channels, but caused
a significant 6-mV hyperpolarizing shift in the voltage-dependence of fast inactivation of
DmNav26 channels (Table 2-1), indicating that TipE has a variant specific effect on the voltagedependence of fast inactivation.
2.4.3. Effect of TipE and TEH1 on the sensitivity of DmNav9-1 channels to deltamethrin
Deltamethrin, a pyrethroid insecticide, induces a slowly decaying tail current associated
with repolarization in voltage clamp experiments (Vais et al. 2000). To determine whether TipE
and TEH1 differentially modulate the activity of deltamethrin, we used a train of depolarizing
pulses to elicit deltamethrin-induced tail current in oocytes expressing DmNav9-1 with or
without TipE or TEH1. At 1 µM, deltamethrin induced a large tail current (Fig. 2-4A), which can
be quantified as the percentage of channel modification by deltamethrin using the method
developed by Tatebayashi and Narahashi (Tatebayashi and Narahashi 1994). Co-expression of
TEH1 with DmNav9-1 channels significantly reduced the percentage of channels modified by
deltamethrin (Fig. 2-4B), whereas TipE had no effect on channel sensitivity to deltamethrin (Fig.
2-4B).
Derst et al. (Derst et al. 2006) reported that the recovery from inactivation of a
Drosophila sodium channel variant was slowed by TEH1. We hypothesized that, in the presence
of TEH1, the trains of depolarizing pulses used in our study to evaluate the effect of deltamethrin
may reduce the availability of open channels, thereby, reducing the gating modification by
deltamethrin. To test whether changes in gating caused by TEH1 were responsible for the
reduced channel sensitivity to deltamethrin, we examined the effect of the multiple depolarizingprepulses on the stability of peak sodium current in channels co-expressed with TipE or TEH1.
36

The peak current remained unchanged in oocytes expressing DmNav9-1 channels alone or with
TipE, whereas the peak sodium current was gradually reduced after each conditioning pulse in
oocytes coexpressing DmNav9-1channels with TEH1 (Fig. 2-4C). These results support the
hypothesis that the reduced deltamethrin sensitivity of DmNav9-1 channels in the presence of
TEH1 is likely caused by reduced availability of open channels. We then examined the effect of
deltamethrin without the conditioning pulses and found that the percentage of channel
modification by deltamethrin was not altered by either TEH1 or TipE (Fig. 2-4D).
2.4.4. Co-expression of TEH1 with DmNav9-1 significantly enhanced entry into and stability of
the fast-inactivated state
The results above prompted us to further characterize the effects of TEH1 and TipE on
inactivation gating kinetics, particularly the development of and recovery from fast-inactivation.
Fig. 2-5A shows the time course of the development of fast inactivation at the pre-pulse voltage
of -45 mV for DmNav9-1 alone or co-expressed with TipE or TEH1. Co-expression of TEH1
with DmNav9-1 greatly enhanced entry of DmNav9-1 channels into the fast inactivated state
compared with that of DmNav9-1 alone or the combination of DmNav9-1 and TipE (Fig. 2-5A).
In addition, the accelerated entry into fast inactivation by TEH1 was observed at all three prepulse voltages tested (Fig. 2-5B).
Co-expressing DmNav9-1 with TEH1 greatly inhibited the recovery of DmNav9-1
channels from fast inactivation all repolarization voltages tested (Fig. 2-5C and D). In contrast,

37

co-expression of DmNav9-1 with TipE had no effect on the recovery from fast inactivation (Fig.
2-5C and D).
2.4.5. Co-expression of TEH1 with DmNav9-1 inhibited recovery from slow inactivation
In addition to fast inactivation, sodium channels undergo slow inactivation which plays
important roles in regulating firing frequency and pattern in response to sustained stimuli (Vilin
and Ruben 2001). Therefore, we examined the effects of TEH1 and TipE on the voltagedependence of slow inactivation, the rate of entry into the slow-inactivated state, and recovery
from slow inactivation of DmNav9-1channels. Co-expression of DmNav9-1 with TEH1 induced
a significant 16 mV hyperpolarizing shift compared with DmNav9-1 alone (Fig. 2-6A and Table
2-1). TEH1 also induced significant hyperpolarizing shifts in voltage-dependence of slow
inactivation in DmNav26 and DmNav22 channels (Table 2-1). In contrast, TipE had no effect on
the voltage-dependence of slow inactivation in any of three variants tested (Table 2-1).
The development of slow inactivation for DmNav9-1 with or without TipE or TEH1 all
exhibited a monophasic time course and was well fitted by an exponential decay (Fig. 2-6B and
Table 2-2). We found that neither TipE nor TEH1 significantly alter the development of slow
inactivation of DmNav9-1 channels (Fig. 2-6B and Table 2-2). The rate of recovery from the
slow inactivated state for DmNav9-1with or without TipE or TEH1 all followed a biphasic time
course (Fig. 2-6C and Table 2-3). DmNav9-1+TipE recovered from slow inactivation in a
manner that was very similar to that of DmNav9-1 alone. However, co-expression with TEH1

38

significantly slowed the slow component (τ2) of recovery and also increased the fraction of the
slow component, but did not alter the fast component (τ1) of recovery (Fig. 2-6C and Table 2-3).
2.5. Discussion
While the roles of mammalian sodium channel β subunits in modulating sodium channel
activities have been extensively studied, research on auxiliary subunits of insect sodium channels
is limited. It is particularly true with respect to how these auxiliary subunits modulate sodium
channel gating and toxin pharmacology. In this study, we showed that while both TipE and
TEH1 enhanced peak sodium currents and increased current decay, they modulated the gating of
DmNav channels differently. First, TEH1 induced hyperpolarizing shifts in the voltagedependences of activation, fast inactivation, and slow inactivation of all three DmNav sodium
channel variants examined. In contrast, TipE did not alter these properties of the three variants,
with one exception: TipE shifted the voltage-dependence of fast inactivation of DmNav26
channels in the hyperpolarizing direction. Second, TEH1, but not TipE, facilitated entry of
sodium channels into fast inactivation and delayed their recovery from both fast and slow
inactivation. Our findings therefore suggest distinct roles of TipE and TEH1 in regulating the
function of sodium channels and neuronal excitability in vivo.
TipE is the first auxiliary subunit of insect sodium channels identified in D.
-

melanogaster. tipE mutants exhibit temperature-sensitive paralytic phenotypes (Feng et al.
1995; Kulkarni and Padhye 1982) suggesting an important role of TipE in regulating neuronal
excitability. An earlier electrophysiological study on the activity of embryonic neurons from a
-

tipE mutant has shown that TipE modulates the activity of only certain neurons (Hodges et al.
39

-

2002). The percentage of embryonic neurons from a tipE mutant capable of firing repetitively
during a sustained depolarization was significantly reduced (Hodges et al. 2002). However, only
-

3

a portion of the tipE neurons was affected (Hodges et al. 2002). Additionally, a [H ]saxitoxin
binding study showed that sodium channel density was reduced by about 30% to 40% in head
-

membrane extracts from the tipE mutants compared with wild type flies (Jackson et al. 1986).
Furthermore, whole-cell path clamp recordings indicated that sodium current density was
-

decreased by about 40% to 60% in dissociated embryonic neurons of tipE mutants (O'Dowd and
Aldrich 1988). Consistent with these findings, TipE was shown to enhance the peak current of
insect sodium channels in heterologous expression (Xenopus oocytes) studies (Dong 2010; Feng
et al. 1995; Warmke et al. 1997).
Although TipE accelerated the current decay of all three variants (Fig. 2-2A-C) in our
study, the effects on these three variants were not as drastic as that on the variant in Warmke et
al., (Warmke et al. 1997), suggesting that the effect of TipE on inactivation kinetics may be
variant-specific. Furthermore, the effects of TipE on sodium channel gating seem to be limited,
compared to TEH1, and may also be variant-specific. The voltage dependence of fast
inactivation of only one variant, DmNav26, was altered in the presence of TipE (Table 2-1).
DmNav26 differs from the other two variants in the exclusion of one optional exon j, and
inclusion of one optional exon f and one mutually exclusive exon k, and also contains five
scattered amino acid changes which are possibly due to RNA editing. Which unique sequence(s)
contributes to this variant-specific effect remains to be determined. It is known that DmNav and
other insect sodium channel transcripts undergo extensively alternative splicing and RNA
40

editing, generating a large collection of sodium channel variants (Dong 2007; Dong 2010). These
variants exhibit unique gating and pharmacological properties (Lin et al. 2009; O'Donnell-Olson
et al. 2008; Song et al. 2006; Song et al. 2004; Tan et al. 2002a). They may be expressed in
different tissues and cells to fulfill their unique roles in insect neurophysiology (Song et al.
2004). It is therefore possible that modulation of gating properties of selective DmNav variants
by TipE provides a unique control of neuronal activities in specific neural circuits. On the other
hand, extensive modification of sodium channel gating properties by TEH1 raises the possibility
that TEH1 plays a broader role than TipE in modulating the gating of potentially diverse sodium
channel variants in Drosophila. Enhanced fast and slow inactivation by TEH1 could lead to
reduced availability of open sodium channels particularly in response to sustained stimulations of
various durations, which could decrease firing frequency and alter firing patterns. As we showed,
reduced availability of open channels by TEH1 decreased the potency of pyrethroids because
pyrethroids preferably act on open sodium channels. It is possible that TEH1, but not TipE, may
regulate sodium channel sensitivity to pyrethroids in neurons that encounter repetitive
stimulations. It is also intriguing that TEH1 could modulate three different gating properties,
activation, fast inactivation, and slow inactivation, which are thought to be controlled by distinct
regions of the sodium channel protein (Catterall 2000; Catterall 2002; Goldin 2003). Further
characterization of how TEH1 modulates these three gating properties at the molecular level may
uncover interconnecting molecular features that are critical for activation, fast inactivation, and
slow inactivation of sodium channels.
Our data suggests that TEH1 has similar modulatory effects on DmNav variants as the β
subunits of mammalian sodium channels. Aside from regulating the expression of sodium
channels, mammalian β subunits modify the gating properties of sodium channels and modulate
41

the electrical excitability of nerves and muscles (Chahine et al. 2008). For example, β1 subunits
induce depolarizing shifts in the voltage-dependence of activation, fast inactivation, and slow
inactivation of Nav1.2 channels (Xu et al. 2007). Additionally, co-expression of β1 subunits with
Nav1.7 and Nav1.8 sodium channels in Xenopus oocytes accelerates current kinetics and
produces a hyperpolarizing shift in steady-state inactivation (Vijayaragavan et al. 2001), and
modulates activation, slow inactivation, and recovery from slow inactivation of Nav1.4 channels
(Vilin et al. 1999; Webb et al. 2009).
In conclusion, we showed that TipE and TEH1 differentially modulate key gating
properties of DmNav, even though both TipE and TEH1 enhance the sodium current and
accelerate current decay in all DmNav variants tested. Furthermore, although TipE and TEH1 are
structurally different from mammalian sodium channel β subunits, our results show that these
proteins appear to be functionally similar. Thus, not only TipE, but also TEH1, may play an
important role in regulating neuronal activities in insects. Further understanding the role of
TEH1 in vivo, including generation and characterization of TEH1 mutants, is expected to further
advance our general knowledge of sodium channel function and neuronal excitability.

42

Table 2-1. Gating properties of DmNav variants with or without TipE or TEH1.
Activation

Fast Inactivation

Slow inactivation

V1/2 (mV)

k (mV)

V1/2 (mV)

k (mV)

V1/2 (mV)

k (mV)

DmNav9-1

-29.0 ± 1.1

5.5 ± 0.7

-41.4 ± 0.8

4.8 ± 0.2

-44.5 ± 1.8

5.6 ± 0.8

+ TipE

-26.2 ± 0.5

5.2 ± 0.2

-42.1 ± 0.7

5.1 ± 0.1

-44.9 ± 1.4

5.5 ± 0.2

+ TEH1

-41.1 ± 1.1* 4.6 ± 0.2

-51.7 ± 0.6*

5.0 ± 0.1

-60.2 ± 0.5*

5.0 ± 0.3

DmNav26

-25.7 ± 0.2

3.4 ± 0.3

-34.1 ± 0.1

4.1 ± 0.2

-45.6 ± 0.2

5.0 ± 0.3

+ TipE

-24.7 ± 0.6

3.4 ± 0.4

-40.8 ± 0.4*

4.2 ± 0.2

-44.1 ± 0.7

4.5 ± 0.4

+ TEH1

-33.6 ± 0.6* 3.1 ± 0.4

-42.6 ± 0.6*

4.6 ± 0.1

-50.6 ± 0.3*

4.5 ± 0.5

DmNav22

-26.7 ± 1.1

5.8 ± 0.5

-40.7 ± 0.6

4.8 ± 0.1

-49.2 ± 0.2

3.7 ± 0.2

+ TipE

-25.1 ± 1.2

7.2 ± 0.5

-38.9 ± 0.5

4.8 ± 0.2

-45.5 ± 0.8

5.3 ± 0.2

+ TEH1

-32.1 ± 0.7* 6.2 ± 0.3

-48.3 ± 0.6*

5.2 ± 0.1

-57.9 ± 0.7*

4.8 ± 0.1

Data represent mean ± SEM for 12-20 oocytes. DmNav9-1, DmNav26, and DmNav22 are treated
as control of each group.
* Significantly different from that of DmNav channel only using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05).

43

Table 2-2. Development of slow inactivation of DmNav9-1 with or without TipE or TEH1
+

Na channel

τ (ms)

f

n

DmNav9-1

3.58 ± 0.81

0.99 ± 0.01

10

+ TipE

5.34 ± 0.43

0.99 ± 0.01

9

+ TEH1

2.37 ± 0.35

0.99 ± 0.01

12

Development of slow inactivation was fitted by an exponential decay function. Data represents
mean ± SEM. τ: time constant, f: fraction of inactivation, n: number of oocytes.

Table 2-3. Recovery from slow inactivation of DmNav9-1 with or without TipE or TEH1
+

Na
channel

τ1

f1

τ2

f2

n

DmNav9-1

0.23 ± 0.02

0.81 ± 0.01

5.10 ± 0.45

0.19 ± 0.01

9

+ TipE

0.24 ± 0.02

0.79 ± 0.01

4.8 ± 0.42

0.21 ± 0.02

8

1
0
Recovery from slow inactivation was fitted by double exponential function. Data represents
mean ± SEM. τ: time constant, f: relative fraction, n: number of oocytes.
+ TEH1

0.24 ± 0.03

0.59 ± 0.02*

7.1 ± 0.38*

0.41 ± 0.01*

*Significant difference compared with DmNav9-1 channel using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05).

44

Figure 2-1. Modulatory effects of TipE and TEH1 on peak sodium currents of DmNav9-1,
DmNav22, or DmNav26 channels.
(A) Representative traces of maximum peak sodium current recorded as introduced bellow from
oocytes expressing DmNav9-1, DmNav9-1+TipE, and DmNav9-1+TEH1 sodium channels. Note
that the sodium current of the DmNav9-1 channel possesses a non-inactivating component,
known as persistent current (10% of the maximal transient peak current). TipE and TEH1 did not
enhance the persistent current. (B) Both TipE and TEH1 significantly increased peak sodium
currents of all three Para sodium variants tested: DmNav9-1, DmNav26, and DmNav22, but there
was no significant difference between the effects of TipE and TEH1. Sodium currents were
recorded 48 hours after cRNA injection. Sodium currents were recorded by a step depolarization
to from -80 to 65 mV in 5mV increments with a holding potential of -120 mV. Data are
presented as mean ± SEM for 12-15 oocytes. * indicates a significant difference compared to
peak of DmNav channel only using one-way ANOVA with Scheffe’s post hoc analysis (p <
0.05).

45

Figure 2-1. (Cont’d)

46

Figure 2-2. Enhancement of sodium current decay by co-expression of TipE or TEH1 with
DmNav channel variants. Sodium current decay in DmNav9-1, DmNav22, or DmNav26 coexpressed with TipE (A, C, and E respectively) or TEH1 (B, D, and F respectively). The decay
of sodium current was fitted by a single exponential to generate time constants of current decay
(τdecay). Each data point represents mean ± SEM for 12-20 oocytes. * indicates a significant
difference compared to that of DmNav channel only (p < 0.05).

47

Figure 2-2. (Cont’d)

48

Figure 2-2 (Cont’d)

49

Figure 2-3. Effects of co-expression of TipE or TEH1 on the voltage-dependence of activation
and fast inactivation of DmNav9-1 channels.
(A) Voltage-dependence of activation. (B) Voltage-dependence of fast inactivation. Data were
fitted with a two-state Boltzmann equation and fitting parameters are shown in Table 2-1. Data
points are shown as mean ± SEM. Recording protocols are indicated and the details of the
protocols and data analysis are described in the Materials and Methods.

50

Figure 2-4. Sensitivity of DmNav9-1 to deltamethrin is modulated by co-expression of TEH1.
(A) A representative tail current induced by 1 µM deltamethrin. (B) Percentage of channel
modification by deltamethrin (multiple-pulse test). Tail currents were elicited by a 66.7-Hz train
of 100 5-ms depolarization from -120 to 0 mV. (C) Co-expression of TEH1 significantly reduced
the stability of DmNav9-1 peak sodium current under repeated conditioning depolarizations.
Sodium currents were recorded during 20-ms step depolarizations from –120 mV to -10 mV after
0-100 conditioning pulses (5-ms pulses from -120 mV to 0 mV at 66.7 Hz). (D) Percentage of
channel modification by deltamethrin (single-pulse test). Tail currents were elicited by a 500 ms
depolarization from -120 mV to 0 mV. All data are shown as mean ± SEM for 9-15 oocytes. *
indicates significant difference compared to the DmNav9-1 channel using one-way ANOVA
with Scheffe’s post hoc analysis (p < 0.05).

51

Figure 2-4. (Cont’d)

52

Figure 2-5. Effects of co-expression of TipE or TEH1 on entry into or recovery from fast
inactivation of DmNav9-1 channels. (A) Time course of development of fast inactivation with a
pre-pulse of -45 mV. (B). τ values of development of fast inactivation under different pre-pulse
potentials. τ values were determined by fitting time course of the development of fast
inactivation with a single exponential decay (n ≥ 12). (C). Recovery from fast inactivation with a
repolarizing potential of -70 mV. (D). τ values of recovery from fast inactivation at different
repolarizing voltages. τ values were calculated by fitting recovery from fast inactivation data
from different repolarizing voltages by a single exponential function (n ≥ 10). Recording
protocols are indicated and the details of the protocols and data analysis are described in the
Materials and Methods.

53

Figure 2-5. (Cont’d)

54

Figure 2-6. Co-expression of TEH1 inhibits recovery from slow inactivation of DmNav9-1
channels. (A) Voltage-dependence of slow inactivation. (B) Time course of development of
slow-inactivation. Development of slow inactivation was fitted by an exponential decay and the
parameters are summarized in Table 2-3. (C) Time course of recovery from slow-inactivation.
Recovery from slow inactivation was fitted by a double exponential function and the parameters
are summarized in Table 2. Recording protocols are indicated and the details of the protocols and
data analysis are described in the Materials and Methods.

55

CHAPTER 3
DISTINCT MODULATING EFFECTS OF TIPE-HOMOLOGOUS PROTEINS 2 AND 4 ON
DROSOPHILA SODIUM CHANNEL SPLICE VARIANTS

56

3.1 Abstract
The Drosophila melanogasdter TipE protein is thought to be an insect sodium channel
auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Four
TipE-homologous genes (TEH1-4) were found in the genome of D. melanogaster. A recent
functional study of TipE and TEH1 revealed common and distinct modulating effects of TipE
and TEH1 on three different Drosophila sodium channel splice variants, but, the relative effects
of the other three TEH proteins on the gating properties of different sodium channel splice
variants remain unknown. In this study, I co-expressed TEH2, TEH3 or TEH4 in Xenopus
oocytes with DmNav9-1, DmNav22, and DmNav26, the three sodium channel splice variants that
were coexpressed previously with TipE or TEH1. TEH2 increased the maximum amplitude of
peak current of all three variants, but did not alter the gating properties of these channels. In
contrast, TEH4 had no effect on peak current, yet caused significant depolarizing shifts in the
voltage-dependence of activation, fast inactivation, and slow inactivation in all three channel
variants. Furthermore, TEH4 induced or enhanced persistent current and slowed sodium current
decay of all three channel variants. TEH3 enhanced an outward current of an unidentified
endogenous channel(s) in Xenopus oocytes, which prevented further analysis of its effects on
DmNav9-1, DmNav22, or DmNav26. This study extends previous findings based on TEH1 and
TipE, and shows both distinct and overlapping effects of the TEH proteins on the gating
properties of different sodium channel splice variants. My findings support the hypothesis that
TipE and the four TEH proteins differentially modulate the gating properties of various sodium
channel variants and constitute an important layer of regulation of membrane excitability in vivo.

57

3.2. Introduction
Voltage-gated sodium channels are responsible for the initiation and propagation of
action potentials in almost all excitable cells. Mammalian sodium channels are composed of a
pore-forming α subunit and one or more β subunits. Sodium channel α subunit is composed of
four homologous domains (I-IV), each containing six transmembrane segments (S1-S6). The S1S4 segments serve as a voltage-sensing module. The S4 segment in each domain contains four to
seven positively charged residues and moves outward in response to membrane depolarization,
which initiates the channel activation process (Catterall 1986; Guy and Seetharamulu 1986). The
S5 and S6 segments and the reentrant loops connecting them compose the inner pore.
Voltage-gated sodium channels change conformation in response to changes in
membrane potential. In response to membrane depolarization, sodium channels open or activate,
and sodium ions flow into the cell. Within a few milliseconds, however, sodium channels close
or inactivate. This fast inactivation is mediated by an inactivation particle formed by residues in
the linker connecting domains III and IV which physically occludes the inner pore preventing
ions from flowing into the cell (Catterall 2012). Under prolonged depolarization, sodium
channels enter into another inactivation state called the slow inactivated state. Unlike fast
inactivation where recovery takes tens of milliseconds, recovery from slow inactivation requires
seconds to minutes of membrane repolatization to return to a resting state (Goldin 2003; Vilin
and Ruben 2001). The processes of sodium channel activation, fast inactivation and slow
inactivation play critical roles in regulating membrane excitability (Goldin 2003).
In mammals, there are at least nine sodium channel isoforms with different gating
properties and expression patterns in various cell types, tissues, and developmental stages
(Catterall 2012; Goldin et al. 2000; Yu and Catterall 2003). In addition, there are four sodium

58

channel β subunits (β1-β4) that have been functionally characterized. The β subunits are small
transmembrane proteins that possess an extracellular immunoglogulin (Ig) domain, a single
transmembrane segment, and a short intracellular C-terminal domain. As sodium channel β
subunits, they modulate gating properties and expression of sodium channels (Patino and Isom
2010). Furthermore, the β subunits function as cell adhesion molecules interacting with both
cytoskeleton and extracellular matrix, regulating cell migration and cellular aggregation
(Brackenbury and Isom 2011; Patino and Isom 2010).
In contrast, insects have only one sodium channel gene that encodes the α subunit
equivalent of mammalian sodium channels (Dong 2010; Loughney et al. 1989), and Drosophila
lacks any orthologs of mammalian β subunits (Littleton and Ganetzky 2000). Instead, alternative
splicing and RNA editing generate a large collection of sodium channel variants which exhibit
diverse gating properties (Dong 2007; Lin et al. 2009; Olson et al. 2008). Additionally TipE is
critical for sodium channel expression and neuronal activities and functions as an auxiliary
-

subunit for insect sodium channels (Feng et al. 1995; Warmke et al. 1997). TipE mutant flies
exhibit a temperature-sensitive paralytic phenotype, similar to sodium channel mutants (Feng et
3

-

al. 1995), and [H ]saxitoxin binding studies of insect embryonic neurons indicate that tipE

mutant flies have reduced sodium channel density (Jackson et al. 1986). Consistently, whole-cell
patch recording indicates that sodium current is decreased about 40% to 60% in dissociated
-

embryonic neurons of tipE mutant flies (O'Dowd and Aldrich 1988). TipE also increases peak
current of sodium channels transiently expressed in Xenopus oocytes (Derst et al. 2006; Feng et
al. 1995; Warmke et al. 1997).

59

In addition to TipE, there are four TipE-homologous genes (TEH1-4) in D. melanogaster
and three to four orthologs in other insect species (Derst et al. 2006). TEH1 is exclusively
expressed in the nervous system, while transcripts of TipE and TEH2-4 are detected in both
neuronal and non-neuronal tissues (Derst et al. 2006). All four TEH proteins differentially
modified one D. melanogaster sodium channel variant expressed in Xenopus oocytes (Derst et al.
2006). A recent study compared the modulation of three different sodium channel splice variants
from D. melanogaster by TipE or TEH1 and revealed that TEH1 extensively modified the
functional properties of all three variants, whereas TipE only modified the gating of one of the
variants (Wang et al. 2013).
Whereas TEH2-4 have been shown to modulate the function of a single sodium channel
variant, the relative effects of TEH2, TEH3, or TEH4 on the gating properties of different
sodium channel splice variants remains unknown. In this study, I examined the modulating
effects of TEH2-4 on gating properties of three D. melanogaster sodium channel variants:
DmNav9-1, DmNav22, and DmNav26 heterologously expressed in Xenopus oocytes. My data
revealed strikingly distinct effects of TEH2 and TEH4 on the expression and function of all three
channel variants. TEH2 enhanced the amplitude of sodium currents without affecting gating, but
TEH4 extensively modified gating properties without affecting current amplitude of all three
variants. Co-expression of TEH3 enhanced an outward current from an unknown endogenous
channel in Xenopus oocytes, which prevented us from further analyzing the modulating effects of
TEH3 on sodium channel gating properties. This study extends previous findings based on TEH1
and TipE, and further shows both distinct and overlapping effects of the TEH proteins on the
gating properties of different sodium channel splice variants, and implies their importance in
regulating insect neuronal excitability in vivo.
60

3.3. Materials and Methods
3.3.1. Xenopus oocyte expression system
Oocytes were obtained surgically from female Xenopus laevis (Nasco, Ft. Atkinson. WI)
2+

and incubated with 1 mg/ml Type IA collagenase (Sigma Co., St. Louis, MO) in Ca -free ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5). Following
digestion, any follicle still attached to the oocytes was physically removed with forceps. Isolated
oocytes were incubated in ND-96 medium supplemented with 1.8 mM CaCl2 supplemented with
50 mg/ml gentamicin, 5 mM pyruvate, and 0.5 mM theophylline (Goldin 1992). Healthy stage
V-VІ oocytes were used for cRNA injection. TEH2-4 cRNA or H2O (as control) was injected
together with DmNav cRNA at a 1:1 ratio.
3.3.2. Electrophysiological recording and analysis
Methods for two-electrode recording and data analysis were similar to those described
previously (Tan et al., 2005). Borosilicate glass electrodes were filled with filtered 3 M KCl in
0.5% agarose and had a resistance of 0.5 to 1.0 MΩ. The recording solution was ND-96 (96 mM
NaCl, 2.0 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES, pH adjusted to 7.5
with NaOH). Sodium currents were measured with a Warner OC725C oocyte clamp amplifier
(Warner Instrument, Hamden, CT) and processed with a Digidata 1440 (Axon Instruments Inc.,
Foster City, CA). Data were sampled at 50 kHz and filtered at 2 kHz. Leak currents were
corrected by p/4 subtraction (Bezanilla and Armstrong 1977). pClamp 10.2 software (Axon
Instruments Inc., CA) and Origin 8.0 software were used for data acquisition and analysis. The
voltage dependence of sodium channel activation was calculated by measuring the peak current

61

at test potentials ranging from
−80 mV to +65 mV in 5

mV increments and divided by (V −

Vrev), where V is the test potential and Vrev is the reversal potential for sodium current. Peak
conductance values were normalized to the maximal peak conductance (Gmax) and fitted with a
−1

two-state Boltzmann equation of the form G/Gmax = [1 + exp (V − V1/2)/k] , in which V is the
potential of the voltage pulse, V1/2 is the voltage for half-maximal activation, and k is the slope
factor.
The voltage dependence of sodium channel fast inactivation was determined by using 100
ms inactivating pre-pulses ranging from -120 mV to 0 mV in 5 mV increments from a holding
potential of −120 mV, followed by test pulses to -10 mV for 20 ms. The peak current amplitude
during the test pulse was normalized to the maximum current amplitude and plotted as a function
of the pre-pulse potential. Data were fitted with a two-state Boltzmann equation of the form
−1

I/Imax = [1 + (exp(V − V1/2)/k)] , in which I is the peak sodium current, Imax is the maximal
current evoked, V is the potential of the voltage pre-pulse, V1/2 is the half-maximal voltage for
inactivation, and k is the slope factor.
The voltage dependence of sodium channel slow inactivation was measured with 60 sec
conditioning pulses ranging from
−100 mV to 0 mV i

n 10 mV increments, followed by

repolarization to a holding potential of -120 mV for 100 ms to remove fast inactivation, and at
last a test pulse of -10 mV for 20 ms. The peak current amplitude during the test depolarization
was normalized to the maximum current amplitude and plotted against the conditioning
potential. Data were fitted with a two-state Boltzmann equation as above for fast inactivation.

62

Development of fast inactivation was measured by holding oocytes at -120 mV, followed
by a pre-pulse to -45 mV for 0 to 80 ms, and then a test pulse of -10 mV for 20 ms to measure
the fraction of sodium current inactivated during the pre-pulse. The peak current during the test
pulse was divided by the peak current which has a pre-pulse duration of 0 ms and plotted as a
function of duration time of pre-pulse. Time constant (τ) was calculated by fitting the plot with a
single exponential decay function.
Recovery from fast inactivation was tested with a conditioning depolarization of -10 mV
for 100 ms, which fast inactivated all sodium channels, then repolarization to -70 mV for 0 - 20
ms followed by a 20 ms test pulse to -10 mV. The peak current during the test pulse was divided
by the peak current during the conditioning pulse and plotted as a function of the duration of the
repolarization pulse. Time constant (τ) of recovery from fast inactivation was calculated by
fitting the plot with a single exponential function.
Development of slow inactivation was measured by holding oocytes at -120 mV,
followed with a -10 mV pre-pulse depolarization for 0 to 25 s, then repolarization to -120 mV for
100 ms to remove fast inactivation, and a test pulse of -10 mV for 20 ms to measure the fraction
of sodium current inactivated during the pre-pulse. The peak current during the test pulse was
divided by the peak current which has a pre-pulse duration of 0 ms and plotted as a function of
duration of pre-pulse. Time constant (τ) was calculated by fitting the plot with an exponential
decay function.
Recovery from slow-inactivation was measured by holding oocytes at -120 mV, followed
by a pre-pulse to -10 mV for 60 s to drive sodium channels into the slow inactivated state,
followed by repolarization to -120 mV for 0 to 30 s, and finally a test pulse to -10 mV for 20 ms.
The peak current during the test pulse was divided by the peak current which has a repolarizing

63

duration of 30 s and plotted as a function of the duration of the repolarization pulse. Recovery
from slow inactivation was well fitted by a double exponential function.
A slow depolarizing ramp protocol from a holding potential of -100 to 20 mV at a ramp
rate of 0.2mV/ms, which promotes inactivation of the rapidly inactivating component of sodium
current, was used to examine the properties of INaP.
3.3.3. Statistical analysis
Results are reported as mean ± SEM. Data were analyzed using one-way analysis of
variance (ANOVA), if ANOVA shows a significant effect, Scheffe’s post hoc comparisons were
used to compare the means. Results were considered to be significant when p < 0.05.
3.4. Results
3.4.1. TEH2 increased the amplitude of peak sodium current of DmNav channels
I examined the effects of TEH2-4 on three splice variants, DmNav9-1, DmNav22, and
DmNav26, expressed in Xenopus oocytes. To determine the effects of TEH2-4 on peak sodium
current, the same amount of cRNA of TEH2, TEH3 or THE4 was co-injected with cRNA of
DmNav into Xenopus oocytes. The amplitude of peak sodium current was measured 48 h after
injection. TEH2 increased the amplitude of peak sodium current of DmNav9-1 channels (Fig. 31A and B), but TEH4 had no effect (Fig 3-1B). Similar results were also observed with THE 2
and 4 for DmNav26 and DmNav22 channels (Fig. 3-1B).
In the course of examining the effects of TEH3 on peak sodium current, I discovered that
TEH3 increased a sustained outward current of an endogenous channel(s) in Xenopus oocytes
(Fig. 3-2C and E). The enhanced outward currents during a 40- ms depolarization from -100 mV
64

to 130 mV from a holding potential of -60 mV was reduced by the application of 1 mM niflumic
2+

-

acid, which is known to inhibit Ca -activated Cl channels in oocytes (Greenwood and Large
1995; Hogg et al. 1994) (Fig. 3-2C, D, and F), but not by a potassium channel blocker (TEA,
data not shown). A majority of oocytes co-expressing sodium channels with TEH3 had
significant sustained outward currents, which prevented me from analyzing the effects of TEH3
on the gating properties of sodium channels. Thus, I focused on TEH2 and TEH4 for the rest of
my experiments.
3.4.2. TEH4 inhibited sodium current decay and increased the persistent current of DmNav
channels.
To determine whether TEH2 and TEH4 modify the rate of fast inactivation, sodium
current decay was calculated by fitting sodium current traces with a single exponential equation.
TEH2 did not affect current decay of any of the three DmNav channels (Figs. 3-3A, B, and C),
but TEH4 significantly slowed sodium current decay of all three variants (Figs. 3-3D, E, and F).

Aside from the transient (i.e., fast inactivating) sodium current (INaT), there is also a noninactivating or persistent current (INaP). The sodium current from the DmNav9-1 sodium channel
contains an INaP component which is about 10% of INaT (Figs. 3-1A, 3-4A and 3-4B). Coexpression of TEH2, TipE or TEH1 with DmNav9-1 did not change the percentage of INaP of the
DmNav9-1 channel, whereas co-expression of TEH4 significantly increased the percentage of
INaP (Fig. 3-4A and B). Similar results were obtained for other two variants: DmNav22 and
DmNav26 (Fig. 3-4C and D).
65

Using a slow voltage ramp, I characterized the gating properties of INaP. The ramp
protocol as introduced in Materials and Methods promotes inactivating INaT and permitting
measurement of INaP. Co-expression of TEH4 with DmNav9-1 resulted in a larger INaP than
expression of DmNav9-1 alone (Fig. 3-5A) without affecting the voltage-dependence of INaP
(Fig. 3-5B).
3.4.3. TEH4 altered the voltage-dependence of activation, fast inactivation, and slow inactivation
of DmNav9-1, DmNav22, and DmNav26.
I determined the modulating effects of TEH2 and TEH4 on voltage-dependent gating
properties of sodium channels. TEH2 did not modify any of the voltage-dependent gating
properties of any of the three variants tested (Table 3-1; Fig. 3-6A, B, and C). TEH4 induced
depolarizing shifts in the voltage-dependence of activation, fast inactivation, and slow
inactivation of all three variants with one exception; co-expression of TEH4 had no effect on the
voltage-dependence fast inactivation of DmNav9-1 channels (Table 3-1; Fig. 3-6A, B, and C).
TEH4 also reduced the voltage-sensitivity of both fast and slow inactivation by increasing the
slope factor of both the fast and slow inactivation curves for all three variants (Table 3-1).
3.4.4. TEH4 altered the kinetics of fast inactivation of DmNav9-1 channels.
Co-expression of TEH4 slowed the entry into and recovery from fast inactivation of
DmNav9-1 sodium channels (Fig. 3-7A-D). In contrast, TEH2 had no effect on the development
of or recovery from fast inactivation of DmNav9-1 channels.

66

TEH1 has been shown to reduce DmNav9-1 sodium channel peak current stability upon
repetitive stimulations (Wang et al., 2013). Therefore, I decided to examine whether the
modulating effect of TEH2 or TEH4 on the peak stability of DmNav9-1 channels. Peak sodium
currents were elicited by a 20 ms depolarization to 0 mV 20 ms after a train of pre-pulses
(ranging from 0-100) from a holding potential of -120 mV to 0 mV for 5 ms at a frequency of
67Hz. Peak current in DmNav9-1 channels was stable under these conditions (e.g. there was no
cumulative inactivation of sodium channels). Co-expression of TEH2 or TEH4 did not reduce
the amplitude of peak current (Fig. 3-8A). These results also suggest that TEH2 and TEH4 may
not alter DmNav9-1 channel kinetics of slow inactivation
Since TEH4 altered the voltage dependence of slow inactivation, I also examined
development of and recovery from slow inactivation with and without TEH2 or TEH4. As
predicted, co-expression of TEH2 or TEH4 did not alter the development of (Fig. 3-8B) or
recovery from slow inactivation (Fig. 3-8C).
3.5. Discussion
In this study, I characterized the effects of TEH2 and TEH4 on peak sodium current and
gating properties of three D. melanogaster sodium channel splice variants, DmNav9-1,
DmNav22, and DmNav26, in Xenopus oocytes. I found that: (1) like TEH1 and TipE (Wang et
al. 2013), TEH2, but not TEH4, increased the amplitude of peak sodium currents of all three
variants; (2) TEH4, but not TEH2 caused depolarizing shifts in the voltage-dependence of
activation, fast inactivation and slow inactivation; (3) TEH4 induced or increased persistent
current and inhibited sodium current decay of all three variants; and 4) TEH4 also altered the

67

kinetics of DmNav9-1 channel fast inactivation. My study extends previous findings based on
TEH1 and TipE, and further shows both distinct and overlapping effects of the TEH proteins on
the gating properties of sodium channel splice variants.
Alternative splicing and RNA editing are two key mechanisms by which insects achieve
the functional diversity of sodium channels. Different splicing types have been reported to
generate sodium channels with distinct gating properties (Lin et al. 2009; Olson et al. 2008).
Furthermore, there is accumulating evidence suggesting that alternative splicing and RNA
editing of sodium channel transcripts contribute to modification of sodium channel function (Lin
et al. 2009; Liu et al. 2004; Song et al. 2004). Specific splicing and RNA editing variants that
have specific functional properties are likely to have distinct tissue/cell expression patterns that
fulfill important roles in the nervous system in vivo. My findings in this study in combination
with Wang et al., (Wang et al. 2013) show that expression of auxiliary subunits in combination
with sodium channel α subunits provides another important mechanism by which insects can
further increase the functional diversity of sodium channels.
Like TEH1, TEH4 caused various modifications of voltage-dependent channel gating and
gating kinetics. However, the functional consequences of the modulations by TEH1 and TEH4
are quite different. First, TEH1 induced hyperpolarizing shifts in the voltage-dependence of
activation, fast inactivation, and slow inactivation (Wang et al. 2013). In contrast, TEH4 induced
depolarizing shifts in the voltage-dependence of all three processes (Table 3-1; Fig. 3-6). Second,
TEH1 accelerated the decay of sodium channel current (Wang et al. 2013), but TEH4 slowed
current decay and also enhanced the persistent (non-inactivating) current that occurs upon
opening of DmNav9-1 channels (Fig. 3-4A, B, C and D). Third, TEH1 significantly accelerated
the entry into fast inactivation and delayed the recovery from fast inactivation, thereby, reducing
68

peak current stability and stabilizing the inactivated state (or promoting the stabilization of the
fast inactivated state). In contrast, TEH4 inhibited both the entry into and recovery from fast
inactivation.
These channel gating processes play distinct physiological roles in regulating neuronal
activities (Catterall 2012; Vilin and Ruben 2001). Voltage-dependent activation is essential for
the initiation of action potentials, while fast inactivation plays an important role in repolarization
of action potential and thus controls the termination of action potential. Slow inactivation is
important in regulating action potential patterns and spike frequency adaptation in response to
repetitive stimuli (Vilin and Ruben 2001). My data suggest that TEH4 may be expressed in cells
where sodium channels remain in the resting state at more depolarized potentials, activate only at
more depolarized potentials, and close more slowly with a persistent current, yet return to the
resting state more quickly so that activation can occur again. TEH1, on the other hand, seems to
be necessary for sodium channels that activate and inactivate more readily and remain
inactivated for a longer period of time.
TEH4 was able to enhance a persistent current (Fig. 3-4A, B, C, and D). Persistent
sodium current is a small fraction of sodium current which maintains sodium ion flux after
inactivation of the classic transient sodium current (Kiss 2008). Persistent sodium currents have
been detected from neurons in various regions of the mammalian brain, such as hippocampal
neurons (Hotson et al. 1979), cortical pyramidal cells (Alzheimer et al. 1993), suprachiasmatic
nucleus (Pennartz et al. 1997), and cerebellar Purkinje neurons (Raman and Bean 1997), as well
as in invertebrate neurons, such as squid giant axons (Clay 2003; Rakowski et al. 2002),
spontaneously active heart interneurons of the leech (Hirudo medicinalis), and aCC motoneurons
in Drosophila (Marley and Baines 2011). This small fraction of sodium channels remain active

69

(open) between action potentials, resulting in sub-threshold depolarization which can contribute
to shaping firing characteristics, boosting synaptic inputs, and generating sub-threshold
membrane oscillations (Crill 1996; Enomoto et al. 2006; Fleidervish and Gutnick 1996; Kole
2011). Mutation-induced persistent sodium currents are associated with epilepsy, ataxia, and
sensitivity to pain (Meisler and Kearney 2005). Also, modulation of persistent currents provides
an excellent substrate for neuronal plasticity. My findings on TEH4 inducing a persistent current
or enhancing existing persistent currents suggest a potential role of TEH4 in controlling firing
frequency in vivo.
TEH2 did not alter any gating properties of any sodium channel tested (Table 3-1; Fig. 36). There are many sodium channel splice variants, and it is possible that TEH2 may have
distinct modulatory effects on specific sodium channel variants, as was seen with TipE (Wang et
al. 2013). TEH2 may play some other as yet undefined regulatory role, such as modulating
sodium channel expression at the plasma membrane.
The role of TEH3 in regulating sodium channel function remains to be determined. In my
experiments, TEH3 enhanced an outward current from an unknown, endogenous ion channel(s)
in Xenopus oocytes. Preliminary experiments, using two ion channel blockers, niflumic acid and
2+

TEA, suggest that the outward current may be a chloride current carried via Ca -activated
chloride channels in Xenopus oocytes (Kuruma and Hartzell 1999). Nevertheless, I was able to
perform functional analysis from a small portion of oocytes (about 3%) expressing TEH3 and
DmNav channels where the TEH3-induced outward current was not so obvious (data not shown).
I found that TEH3: (1) induced depolarizing shifts in voltage dependence of activation and
inactivation; (2) inhibited sodium current decay; (3) and increased persistent sodium currents.
My findings implicate a potential role of TEH3 in modulating sodium channel functions and also
70

2+

+

the functions of other ion channels, such as big-conductance Ca -activated K channels, which
has been predicted to be a potential partner for TEH3 and TEH4 (Derst et al. 2006) based on
sequence similarity.
The distinct modulating effects of TipE, TEH1, TEH2, and TEH4 on channel expression
and gating properties of DmNav channels resemble the effects of β subunits on mammalian
sodium channels. Different mammalian β subunits have been shown to differentially modulate
expression and gating properties of mammalian sodium channels expressed in Xenopus oocytes.
For example, β1 and β2 subunitsincrease peak sodium current of Nav1.2 channels transiently
expressed in Xenopus oocytes (Isom et al. 1992; Isom et al. 1995), and β3 subunits increase peak
sodium current of Nav1.5 and Nav1.8 in Xenopus oocytes (Fahmi et al. 2001; Morgan et al.
2000). β2 causes a hyperpolarizing shift in the voltage-dependence of activation of Nav1.6, but
β1 does not (Tan and Soderlund 2011). The β1 subunit was shown to cause a negative shift in
voltage-dependence of inactivation of Nav1.2 channels, however (Isom et al. 1992). β3, but not
β1 or β2, increases the persistent sodium current of Nav1.2 expressed in HEK293 cells (Qu et al.
2001).
In mammals, specific sequences or domains of β subunits are also associated with
specific functions. The extracellular N-terminus or intracellular C-terminus domains of β
subunits have been shown to mediate the modulation of sodium channel gating properties (Chen
and Cannon 1995; McCormick et al. 1998; Zhao et al. 2011). The sequence features or protein
domains that are critical for the modulatory effects of TipE and TEH proteins on sodium
channels remain to be determined.
71

In conclusion, my results show that TEH2-4 proteins each have distinct modulatory
effects on sodium channel expression and gating properties. The findings in this study and an
earlier study (Wang et al. 2013) provide evidence supporting the notion that these TEH proteins
function as auxiliary subunits of insect sodium channels just as β subunits for mammalian
sodium channels. TEH1-4 proteins likely constitute an additional layer of regulation of
membrane excitability in vivo.

72

Table 3-1. TEH1, TEH2 and TEH4 modulated voltage-dependence of activation, fast
inactivation, and slow inactivation of DmNav9-1, DmNav26, and DmNav22 expressed in
Xenopus oocytes.
Activation
Fast Inactivation
Slow inactivation
V1/2 (mV)

k

V1/2 (mV)

k

V1/2 (mV)

k

DmNav9-1

-30.7 ± 1.1

5.4 ± 0.7

-39.2 ± 0.5

4.9 ± 0.2

-44.9 ± 1.6

5.3 ± 0.8

+TEH1

-41.1 ± 1.1*

4.6 ± 0.2

-51.7 ± 0.6*

5.0 ± 0.1

-60.2 ± 0.5* 5.0 ± 0.3

+TEH2

-29.4 ± 1.1

4.2 ± 0.3

-37.8 ± 0.5

4.2 ± 0.1

-44.5 ± 0.5

+TEH4

-17.0 ± 1.2*

4.8 ± 0.2

-34.4 ± 0.9*

5.8 ± 0.2*

-35.8 ± 1.0* 6.6 ± 0.5*

DmNav26

-27.3 ± 0.5

3.5 ± 0.3

-35.4 ± 0.1

4.4 ± 0.2

-47.6 ± 0.9

+TEH1

-33.6 ± 0.6*

3.1 ± 0.4

-42.6 ± 0.6*

4.6 ± 0.1

-50.6 ± 0.3* 4.5 ± 0.5

+TEH2

-26.2 ± 1.0

3.5 ± 0.2

-36.5 ± 0.5

4.3 ± 0.1

-44.3 ± 0.3

+TEH4

-17.7 ± 1.7*

4.1 ± 0.3

-32.1 ± 0.1

5.9 ± 0.2*

-37.6 ± 1.2* 7.7 ± 0.4*

DmNav22

-27.2 ± 1.2

5.3 ± 0.4

-41.5 ± 0.5

4.7 ± 0.1

-49.8 ± 0.2

+TEH1

-32.1 ± 0.7*

6.2 ± 0.3

-48.3 ± 0.6*

5.2 ± 0.1

-57.9 ± 0.7* 4.8 ± 0.1

+TEH2

-24.9 ± 1.0

5.0 ± 0.2

-36.3 ± 0.4

4.3 ± 0.1

-45.7 ± 0.3

+TEH4

-21.6 ± 1.3*

5.6 ± 0.3

-35.8 ± 0.4*

5.6 ± 0.3*

-40.7 ± 1.5* 5.5 ± 0.2*

4.8 ± 0.2

4.4 ± 0.5

3.5 ± 0.4

4.7 ± 0.5

4.2 ± 0.1

Data represent mean ± SEM for 9-18 oocytes. DmNav9-1, DmNav26, and DmNav22 are treated
as control of each group. * Significant difference compared with DmNav channels using oneway ANOVA with Scheffe’s post hoc analysis (p<0.05). Results of TEH1 have been reported in
a previous study (Wang et al., 2013).

73

Figure 3-1. Effects of TEH2 and TEH4 on peak sodium currents of DmNav channels. (A)
Representative traces of maximum peak sodium currents recorded as introduced bellow from
oocytes expressing DmNav9-1, DmNav9-1 + TEH2, and DmNav9-1 + TEH4 channels. (B)
TEH2 and TEH4 differentially modulate peak sodium current of DmNav9-1, DmNav22 and
DmNav26 in Xenopus oocytes. Sodium currents were recorded 48 hours after cRNA injection.
Sodium currents were recorded by a step depolarization to -80 to 65 mV in 5mV increments
from a holding potential of -120 mV. Data are presented as mean ± SEM for 10-18 oocytes. *
indicates a significant difference compared to peak current of DmNav9-1 channel using a oneway ANOVA with Scheffe’s post hoc analysis (p < 0.05).

74

Figure 3-1. (Cont’d)

75

Figure 3-2. Expression of TEH3 alone in Xenopus oocytes induced a niflumic acid sensitive
outward current. Outward current was recorded by 40 msec depolarization from -100 mV to 130
mV with a holding potential of -60 mV. The outward current was recorded three days after
injection. (A) & (B) Outward current from oocyte injected with water before and 10 min after
niflumic acid application, respectively. (C) & (D) Outward current from TEH3 expressed oocyte
before and 10 min after niflumic acid application, respectively. (E) Amplitude of outward current
at different depolarizing voltages ranging from -100 mV to 130 mV without niflumic acid
application. (F) Amplitude of outward current from oocytes with or without TEH3 injection at
the depolarizing voltage of 130 mV before and after niflumic acid application. Data are shown as
mean ± SEM for 6-11 oocytes in E and F. * indicates significant difference using one-way
ANOVA with Scheffe’s post hoc analysis (p < 0.05).

76

Figure 3-2 (cont’d)

77

Figure 3-2. (Cont’d)

78

Figure 3-3. TEH4, but not TEH2, inhibited the current decay of DmNav channels.
(A) & (B) & (C) TEH2 had no effect on sodium current decay of any of the three DmNav
channels. (D) & (E) & (F) TEH4 inhibited sodium channel decay of all three DmNav channels.
The decay of sodium current was fitted by a single exponential to generate the time constants of
current decay (τdecay). Data are presented as mean ± SEM for 10-20 oocytes. * indicates a
significant difference compared to that of DmNav channels only (p < 0.05).

79

Figure 3-3 (cont’d)

80

Figure 3-3. (Cont’d)

81

Figure 3-4. Co-expression of TEH4 increased persistent sodium current of DmNav channels
expressed in Xenopus oocytes. (A) Representative peak current traces of DmNav9-1, or coexpression with TipE, TEH1-2, and TEH4. (B), (C), & (D) Modulating effects of TipE or TEH
proteins on persistent current of DmNav9-1, DmNav22, or DmNav26 channels, respectively. All
data are shown as mean ± SEM for 9-18 oocytes. * indicates significant difference compared to
the DmNav channels using one-way ANOVA with Scheffe’s post hoc analysis (p < 0.05).

82

Figure 3-4 (cont’d)

83

Figure 3-5. Effects of TEH4 on the persistent current of DmNav9-1 channel under ramp protocol
test. (A) Representative traces of persistent current. (B) Normalized persistent sodium current at
different voltages. Amplitude of persistent currents was normalized to the amplitude of peak of
transient sodium current. All data are shown as mean ± SEM for 9-12 oocytes. * indicates
significant difference compared to the DmNav9-1 channel using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05).

84

Figure 3-6. Effects of co-expression of TEH2 and TEH4 on the voltage-dependence of
activation, fast inactivation, and slow inactivation of DmNav9-1 channels. (A) Voltagedependences of activation. (B) Voltage-dependence of fast inactivation. (C) Voltage-dependence
of slow inactivation. Data were fitted with a two-state Boltzmann equation and fitting parameters
are shown in Table 3-1. Data points are shown as mean ± SEM. Recording protocols are
indicated and the details of the protocols and data analysis are described in Materials and
Methods.

85

Figure 3-6. (Cont’d)

86

Figure 3-7. Effects of co-expression of TEH2 or TEH4 on entry into or recovery from fast
inactivation of DmNav9-1 channels. (A) Time course of development of fast inactivation with a
pre-pulse of -45 mV. (B) τ values of development of fast inactivation under different pre-pulse
voltages. τ values were determined by fitting time course of the development of fast inactivation
with a single exponential decay (n > 10). (C) Recovery from fast inactivation with a repolarizing
voltage of -70 mV. (D) τ values of recovery from fast inactivation at different repolarizing
voltages. τ values were calculated by fitting recovery from fast inactivation data from different
repolarizing voltages by a single exponential function (n > 9).

87

Figure 3-7 (Cont’d)

88

Figure 3-8. Neither TEH2 nor TEH4 altered the kinetics of slow inactivation of DmNav9-1
channels. (A) Neither TEH2 nor TEH4 altered the peak stability of DmNav9-1 channels; (B)
Development of slow inactivation or (C) Recovery from slow inactivation. Protocols for testing
peak stability, development of slow inactivation, and recovery from slow inactivation are
described in Materials and Methods.

89

Figure 3-8. (Cont’d)

90

CHAPTER 4
THE N1575Y MUTATION IN THE SODIUM CHANNEL OF ANOPHELES GAMBIAE
SYNERGIZES THE EFFECT OF L1014F/S MUTATIONS ON PYRETHROID RESISTANCE

91

4.1 Abstract
Pyrethroid insecticides possess high insecticidal activities and low mammalian toxicity
and represent one of the most powerful weapons in the global fight against malaria and other
arthropod-borne human diseases. However, pyrethroid resistance has become a serious obstacle
to effective use of pyrethroids in controlling these human disease vectors. One major mechanism
of pyrethroid resistance, known as knockdown resistance (kdr), is caused by mutations in the
sodium channel, which is the target site of pyrethroid action. In the African malaria vector
Anopheles gambiae, two kdr mutations, L1014F/S, are found in many pyrethroid resistant
populations globally. Recently, a new sodium channel mutation N1575Y was found to be
concurrent with the L1014F mutation in several pyrethroid resistant populations of An. gambiae.
To examine the role of this new sodium channel mutation in pyrethroid resistance, I introduced
the N1575Y mutation into an Aedes aegypti sodium channel, AaNav1-1, either alone or in
combination with the L1014F mutation, and examined the gating properties of the mutant
channels expressed in Xenopus oocytes. My analyses showed that the N1575Y mutation, by
itself, did not alter AaNav1-1 channel sensitivity to two pyrethroid insecticides: permethrin and
deltamethrin. However, when mutated together with L1014F, the N1575Y mutation made
L1014F channels 10-fold and 4-fold more resistant to permethrin and deltamethrin respectively.
The N1575Y mutation also synergized the effect of another kdr mutation, L1014S, which is
widely distributed in pyrethroid-resistant An. mosquitoes. None of the mutations I tested
significantly affected channel gating. My findings demonstrated that N1575Y functions as an
enhancer of the L1014F/S-mediated pyrethroid resistance and provided a molecular explanation
for the emerging co-occurrence of N1575Y and L1014F in pyrethroid-resistant populations.

92

4.2. Introduction
Voltage-gated sodium channels are transmembrane proteins responsible for the rapidly
rising phase of action potentials and are critical for electrical signaling in most excitable cells
(Catterall 2012). The pore-forming α-subunit of the sodium channel is composed of four
homologous domains (I-IV), each formed by six transmembrane segments (S1-S6) that are
connected by intracellular and extracellular loops. The S1-S4 segments serve as the voltagesensing module, whereas the S5 and S6 segments and the loops connecting them function as the
pore-forming module. In response to depolarization, S4s move outward, then sodium channel
open, which results in sodium ion influx and further depolarization of the membrane potential.
This process is called channel activation (Catterall 2000). After channel opening, sodium channel
inactivate rapidly, within a few milliseconds, in a process known as fast inactivation (Catterall
2000). In response to repolarization, sodium channels go back to resting state in a process of
deactivation.
Because of their critical role in membrane excitability, sodium channels are a primary
target site for a variety of neurotoxins, including pyrethroid insecticides (Catterall 1986;
Narahashi 1987; Soderlund et al. 2002). Pyrethroids preferentially bind to activated sodium
channels, inhibit channel inactivation and deactivation, and result in the prolonged opening of
sodium channels (Narahashi 1996; Vijverberg et al. 1982).
Due to their high insecticidal activity and relatively low mammalian toxicity, pyrethroid
insecticides are widely used to control agricultural pests and diseases vectors. Insecticide-treated
nets (ITNs) are important tools used to protect people from mosquitoes, including An. gambiae
and A. aegypti mosquitoes, which are well known vectors of malaria and dengue respectively
(Mansotte et al. 2010; Willey et al. 2012). Currently, only pyrethroid insecticides are

93

recommended by the World Health Organization for ITNs. However, intensive use of
pyrethroids has led to the selection of pyrethroid resistance in many insect populations around
the world. Pyrethroid resistance has become a serious obstacle to effective mosquito control. One
major mechanism of pyrethroid resistance is knockdown resistance (kdr), which is the result of
mutations in the sodium channel and which causes reduced neuronal sensitivity to DDT and
pyrethroids (Dong 2007; Soderlund 2008). The most common kdr mutation is a leucine to
phenylalanine (L1014F) in IIS6, a mutation what is also detected in the malaria vector Anopheles
mosquito species in many regions around the world (Chen et al. 2008a; Enayati et al. 2003;
Ranson et al. 2000; Singh et al. 2010; Syafruddin et al. 2010; Verhaeghen et al. 2010). Several
other sodium channel mutations were also reported in different mosquito species. These
mutations included: leucine to serine or tryptophan at position 1014 in Anopheles mosquitoes
(Ranson et al. 2000; Tan et al. 2012), valine to glycine or isoleucine at position 1016 in Aedes
mosquitoes (Brengues et al. 2003; Saavedra-Rodriguez et al. 2007), and phenylalanine to cystein
at the position 1534 in Aedes mosquitoes (Kasai et al. 2011; Kawada et al. 2009).
Recently, a new sodium channel mutation N1575Y was reported in An. gambiae
mosquito in Africa (Jones et al. 2012). Interestingly, the N1575Y mutation was always detected
together with L1014F and no mosquitoes were detected harboring only the N1575Y mutation
(Jones et al. 2012). Pyrethroids bio-assays indicated that mosquitoes carrying double mutations
(N1575Y+L1014F) were more resistant to permethrin than mosquitoes carrying only L1014F
(Jones et al. 2012). However, whether the N1575Y mutation confers pyrethroid resistance has
not yet been functionally confirmed.
So far, no sodium channels from Anopheles gambiae have been successfully expressed in
Xenopus oocyte expression systems for functional characterization. In this study, I took

94

advantage of the recent establishment of a Xenopus oocyte expression system for the first
mosquito sodium channel, AaNav1-1 from Aedes aegypti, to investigate the role of N1575Y in
pyrethroid resistance. My results indicated that the N1575Y mutation alone does not alter sodium
channel gating or sensitivity to pyrethroids, but it does exhibit a synergistic effect on L1014Finduced resistance to pyrethroids.
4.3. Materials and Methods
4.3.1. Site-directed mutagenesis
The mosquito Aedes Aegypti sodium channel AaNav1-1 was used as a backbone of all
mutants used in this study. All mutant sodium channels reported in this paper were constructed
using site-directed mutagenesis. Site-directed mutagenesis was performed by PCR using mutant
primers and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). All mutagenesis results
were confirmed by DNA sequencing.
4.3.2. Expression of AaNav sodium channels in Xenopus oocytes
The procedures for oocyte preparation and cRNA injection are identical to those
described previously (Tan et al. 2005). For robust expression of AaNav sodium channels, cRNA
was coinjected into oocytes with Aedes Aegypti TIPE cRNA (1:1 ratio), which enhances the
expression of insect sodium channels in oocytes.
4.3.3. Electrophysiological recording and analysis
The voltage dependence of activation and inactivation was measured using the twoelectrode voltage clamp technique. Methods for two-electrode recording and data analysis were
identical to those described previously (Tan et al. 2005).

95

The voltage dependence of sodium channel conductance (G) was calculated by
measuring the peak current at test potentials ranging from -80 mV to +65 mV in 5 mV
increments and divided by (V – Vrev), where V is the test potential and Vrev is the reversal
potential for sodium current. Peak conductance values were normalized to the maximal peak
conductance (Gmax) and fitted with a two-state Boltzmann equation of the form G/Gmax = [1 +
-1

exp(V-V1/2)/k] , in which V is the potential of the voltage pulse, V1/2 is the voltage for halfmaximal activation, and k is the slope factor.
The voltage dependence of sodium channel inactivation was determined by using 100-ms
inactivating prepulses ranging from -120 mV to 10 mV in 5 mV increments from a holding
potential of -120 mV, followed by test pulses to -10 mV for 20 ms. The peak current amplitude
during the test depolarization was normalized to the maximum current amplitude and plotted as a
function of the prepulse potential. Data were fitted with a two-state Boltzmann equation of the
-1

form I/Imax = [1 + (exp (V - V1/2)/k)] , in which I is the peak sodium current, Imax is the
maximal current evoked, V is the potential of the voltage prepulse, V1/2 is the half-maximal
voltage for inactivation, and k is the slope.
4.3.4. Measurement of tail currents induced by pyrethroids
The method for application of pyrethroids in the recording system was identical to that
described by Tan et al. (Tan et al. 2005). The effects of pyrethroids were measured 10 min after
toxin application. The pyrethroid-induced tail current was recorded during a 100-pulse train of 5
ms step depolarizations from – 120 to 0 mV with 5 ms interpulse intervals (Vais et al., 2000).
The percentage of channels modified by pyrethroids was calculated using the equation M =
{[Itail/(Eh – ENa)]/[INa/(Et – ENa)]} X 100 (Tatebayashi and Narahashi 1994), where Itail is the
96

maximal tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is
the reversal potential for sodium current determined from the current-voltage curve, INa is the
amplitude of the peak current during depolarization before pyrethroid exposure, and Et is the
potential of step depolarization. Concentration-response curves were fitted by the Hill equation:
n

M = Mmax/{1 + (Kd/[P]) }, in which [P] represents the concentration of pyrethroid, Kd
represents the concentration of pyrethroid that produced the half-maximal effect, n represents the
Hill coefficient, and Mmax is the maximal percentage of sodium channel modified.
4.3.5. Chemicals
Deltamethrin was kindly provided by Bhupinder Khambay (Rothamsted Research,
Harpenden, UK). Deltamethrin was dissolved in dimethyl sulfoxide (DMSO). The working
concentration was prepared in DN96 recording solution immediately prior to experiments. The
concentration of DMSO in the final solution was <0.5%, which had no effect on the function of
sodium channels.
4.3.6. Statistical analysis
Data were sampled at 50 kHz and filtered at 2 kHz. Leak currents were corrected by p/4
subtraction. pClamp 10.2 software (Axon Instruments Inc., CA) and Origin 8.0 software were
used for data acquisition and analysis. Results are reported as mean ± SEM. Data were analyzed
using one-way analysis of variance (ANOVA), if ANOVA shows a significant effect, Scheffe’s
post hoc comparisons were used to compare the means. Results were considered to be significant
when p < 0.05.

97

4.4. Results
4.4.1. N1575Y did not alter the current amplitude or gating properties of the AaNav1-1 channel
The N1575Y mutation is located in the linker that connects domains III and IV (Fig. 4-1).
Amino acid sequence, MFMT, in this linker has been shown to be critical for channel fast
inactivation (Fig. 4-1) (Goldin 2003). To determine whether the N1575Y mutation alters the
inactivation kinetics, I introduced this mutation into AaNav1-1 by site-directed mutagenesis and
expressed both AaNav1-1 and mutant channels in Xenopus oocytes. Both AaNav1-1 and
N1575Y channels produced sodium currents (Fig. 4-2A). Sodium current decay was analyzed
by fitting the decaying part of current traces with a single exponential function. AaNav1-1 and
N1575Y channels had similar rates of decay over the entire range of voltages examined,
indicating that the mutation did not alter fast inactivation kinetics (Fig. 4-2B). Furthermore,
N1575Y mutation did not alter the voltage-dependence of activation or inactivation (Table 4-1
and Fig. 4-3A and C).
Since the N1575Y mutation was detected along with the L1014F mutation in Anopheles
gambiae (Jones et al. 2012), the N1575Y change was introduced into AaNav1-1 carrying the
L1014F mutation, which was available from another study (Du et al., 2013), to generate the
double mutation construct N1575Y+L1014F by site-directed mutagenesis. L1014F and
N1575Y+L1014F channels exhibited similar fast inactivation kinetics compared with the
AaNav1-1 channel (Fig. 4-2B). The voltage-dependence of activation and inactivation of both
channels were also similar to those of the AaNav1-1 channel (Table 4-1, Fig. 4-3A, and C).

98

4.4.2. N1575Y had no effect on AaNav1-1 channel sensitivity to pyrethroids but enhanced
L1014F-mediated resistance to pyrethroids
To examine the effect of the N1575Y mutation on AaNav1-1 channel sensitivity to
pyrethroids, I compared AaNav1-1, N1575Y, L1014F and L1014Y+N1575Y channels’
sensitivities to two pyrethroids: a type I pyrethroid, permethrin, and a type II pyrethroid,
deltamethrin. The percentage of sodium channel modification by pyrethroids was determined by
measuring pyrethroid-induced tail currents upon repolarization in voltage-clamp experiments
(Fig. 4-4A, B, C, and D). Fig. 4-4A shows a tail current induced by 1.0 µM permethrin on
AaNav1-1 channels. The effect of permethrin on N1575Y channels was similar to that of
AaNav1-1 channels. However, the permethrin-induced tail current was significantly reduced by
the L1014F mutation and more drastically reduced by the L1014Y+N1575Y double mutation
(Fig. 4-4B-D). The dose response curves of the wild-type and three mutant channels for
permethrin and deltamethrin are presented in Fig. 4-4E and 4-4F respectively. The N1517Y
channel was as sensitive to both pyrethroids as the AaNav1-1 channel. As expected, the L1014F
mutant channel was about 8-fold more resistant to permethrin than the AaNav1-1 channel (Fig.
4-4E). Similarly, the L1014F channel was 14-fold more resistant to deltamethrin (Fig. 4-4F).
Remarkably, the L1014F+N1575Y channel was 80-fold more resistant to permethrin and 53-fold
more resistant to deltamethrin. Therefore, the N1575Y mutation contributed 9.8-fold and 3.4fold resistance, respectively, to permethrin and deltamethrin in the N1575Y+L1014F channel.
4.4.3. N1575Y enhanced the L1014S-mediated resistance to pyrethroids

99

Another kdr mutation at amino acid position 1014, L1014S, was detected in both
Anopheles and Culex species of mosquitoes (Ranson et al. 2000); Martinez-Torres et al., 1999;).
To determine whether the N1575Y mutation also enhances the effect of L1014S on pyrethroid
action, I made a N1575Y+L1014S double-mutation construct. Like the L1014F mutation, the
L1014S channel was 6.7-fold and 9-fold more resistant to permethrin and deltamethrin
respectively, than the AaNav1-1 channel (Fig. 4-5A and B). The N1575Y+L1014S double
mutation channel was 58-fold and 32-fold more resistant to permethrin and deltamethrin
respectively (Fig. 4-5A and B). In addition, the L1014S and N1575Y+L1014S channels were
not altered in voltage-dependence of activation and inactivation (Table 4-1, Figure 4-3B and D).
4.4.4. N1575Y has no effect on the V1016G-mediated resistance to pyrethroids
My results above clearly indicate that the N1575Y mutation has a synergistic effect on
pyrethroid resistance caused by L1014F/S mutations. To see whether such synergism extends to
pyrethroid resistance caused by other kdr mutations, I included another kdr mutation, V1016G,
in our study. The V1016G mutation is also located in IIS6, two amino acids downstream of
L1014F/S, and has been detected in pyrethroid resistant populations of A. aegypti (SaavedraRodriguez et al. 2007). I introduced the N1575Y mutation into the V1016G channel construct,
which was available from a previous study (Du et al. 2013). Consistent with the finding from the
previous study, the V1016G mutation significantly reduced AaNav1-1 channel sensitivity to both
permethrin and deltamethrin (Fig. 4-6A and B). However, the addition of the N1575Y mutation
did not further enhance the level of resistance to pyrethroids (Fig. 4-6A and B).
4.5. Discussion
In this study, I characterized a novel pyrethroid-resistance-associated mutation N1575Y
in an Aedes sodium channel expressed in Xenopus oocytes. Our study showed that: (1) the
100

N1575Y mutation by itself did not alter sodium channel gating and sensitivity to pyrethroids; (2)
the kdr mutations L1014F/S significantly reduced channel sensitivity to pyrethroids; and (3)
introduction of N1575Y into the L1014F/S channels increased channel resistance to pyrethroids
without modifying channel gating properties. These results suggest that the N1575Y mutation in
the presence of the kdr mutation L1014F is responsible for the high levels of pyrethroid
resistance observed in mosquito populations.
I found that the N1575Y mutation did not alter sodium channel gating properties. In
contrast, the L1014F mutation has been reported to cause depolarizing shifts in voltagedependence of activation and fast inactivation of Musca domestica and Drosophila melanogaster
sodium channels expressed in Xenopus oocytes (Burton et al. 2011; Lee et al. 1999). The L1014S
mutation induced a depolarizing shift in voltage-dependence of inactivation (Burton et al. 2011).
However, other studies showed that the L1014F mutation did not cause significant shifts in
voltage-dependence of activation or inactivation in sodium channels of Blattella germanica and
D. malanogaster expressed in Xenopus oocytes (Tan et al. 2002a; Vais 2000). My results were
consistent with the later studies.
The linker connecting sodium channel domains III and IV is important for sodium
channel fast inactivation (Catterall 2002; Goldin 2003). Besides N1575Y, another sodium
channel mutation, L1596P, within the linker connecting domain III and IV in varroa mites has
been reported to be associated with pyrethroid resistance (Fig. 4-1) (Liu et al. 2006). It is not
clear how amino acids located in the intracellular linker and outside pyrethroid binding sites
modify sodium channel sensitivity to pyrethroids. I speculate that some amino acid residues
located in this linker modify channel sensitivity to pyrethroids by allosterically interacting with
kdr-associated amino acid residues located elsewhere in the sodium channel.

101

Two pyrethroid binding sites have been reported based on computer modeling and
electrophysiological study. The first pyrethroid receptor was proposed in the open state of the
house fly sodium channel based on the crystal structure of voltage-gated potassium channel
(O'Reilly et al. 2006). This model indicated that pyrethroids bind to a triangle pocket composed
of the linker connecting S4 and S5 in domain II, domain II S5, and domain III S6. The second
pyrethroid receptor was built on a mosquito sodium channel based on the crystal structure of a
potassium channel and a bacterial sodium channel (Du et al. 2013). This second pyrethroid
receptor was composed of the linker connecting S4 and S5 in domain I, domain I S5, and domain
II S6 (Du et al. 2013).
Based on the modeling information, the kdr mutation L1014F in IIS6 is located within
the second pyrethroid receptor site, while another kdr mutation V1016G is located within the
first receptor site (Du et al. 2013). The N1575Y mutation increased L1014F/S-induced resistance
to pyrethroids but had no effect on V1016G-induced resistance to pyrethroids. This indicated that
the role of the N1575Y mutation in increasing pyrethroid resistance is site 2-specific. N1575Y
could allosterically reduce the pyrethroid binding by shifting the helices S6 in domain I or
domain II, thereby enhancing pyrethroid resistance caused by L1014F/S mutations. However, the
pyrethroid resistance caused by the V1016G mutation (which is also located in IIS6, but in site
1) was not affected by N1575Y. This may be because glycine, with only a hydrogen atom in the
side chain, can tolerate a small distortion by the N1575Y mutation. Further experiments will be
needed to explain the detailed mechanism of how N1575Y specifically increases L1014F/S
induced pyrethroid resistance.
A similar situation was reported in another study with two sodium channel mutations
(E435K and C785R) located in the linker connecting domains I and II (Tan et al. 2002a). Neither

102

of them alone reduced sodium channel sensitivity to pyrethroids, however, either one concurrent
with the kdr mutation L1014F significantly increased pyrethroid resistance. These N1575Y-,
E435K-, and C785R-associated mechanisms of increased resistance to pyrethroids appeared to
be different with the effect of the super-kdr mutation M918T, located in the linker connecting S4
and S5 in domain II, which is also found to be associated with the L1014F mutation in the field
(Lee et al. 1999). Specifically, M918T alone could reduce sodium channel sensitivity to
pyrethroids, while a higher level of resistance was detected when M918T was combined together
with another kdr mutation, such as L1014F (Vais 2000). Interestingly, both N1575Y and the
super-kdr M918T alone are not known to exist in nature except in association with L1014F kdr
mutation (Jones et al. 2012; Lee et al. 1999).
In conclusion, my results confirmed that the N1575Y mutation exhibited synergistic
effects with the kdr mutation L1014F/S in terms of resistance to pyrethroids. The selective
advantage of evolving the N1575Y mutation to confer high-level pyrethroid resistance is
dependent on the preexistence of the L1014F mutation.

103

Table 4-1. Voltage-dependence of activation and fast inactivation of AaNav1-1 and its mutant
channels
Activation
Fast Inactivation
V1/2 (mV)

k (mV)

V1/2 (mV)

k (mV)

n

AaNav1-1

-33.6 ± 1.1

5.7 ± 0.3

-53.7 ± 0.6

5.0 ± 0.1

19

N1575Y

-32.3 ± 1.6

6.6 ± 0.3

-56.5 ± 0.5

5.0 ± 0.1

20

L1014F

-31.3 ± 0.9

4.0 ± 0.3

-49.6 ± 0.4

4.5 ± 0.1

10

L1014S

-36.3 ± 1.1

4.3 ± 0.3

-50.0 ± 0.3

4.7 ± 0.1

11

N1575Y+L1014F

-28.6 ± 1.1

5.6 ± 0.2

-51.1 ± 0.4

4.8 ± 0.1

27

N1575Y+L1014S

-33.8 ± 1.2

5.1 ± 0.4

-51.5 ± 0.7

4.9 ± 0.1

10

The voltage-dependence of activation and inactivation data were fitted with two-state Boltzmann
equations, as described in Materials and Methods, to determine V1/2, the voltage for halfmaximal conductance or inactivation, and k, the slope for conductance or inactivation. Each
value represents the mean ± SEM.

104

Figure 4-1. The positions of the pyrethroid resistance-associated mutations relevant to this study.
The sodium channel transmembrane protein consists of four homologous domains (I-IV), each
formed by six transmembrane segments (S1-S6) connected by intracellular and extracellular
loops. Residue positions correspond to the housefly sodium channel (GenBank accession
number: AAB47604 and AAB47605). Positions in parentheses correspond to AaNav sodium
channel (GenBank accession number: EU399181). L1014F/S mutations were detected in many
pyrethroid resistant populations in Anopheles gambiae (Rinkevich et al. 2013), and N1575Y was
detected in West and Central Africa populations of Anopheles gambiae (Jones et al., 2012). The
V1016G mutation was found in Aedes aegypti in many regions of the world (Rinkevich et al.,
2013). The entire amino acid sequence of the linker connecting domains III and IV is shown
below the topology diagram. The MFMT motif that is critical for fast inactivation is boxed. The
N1575Y mutation is marked in bold. A previously confirmed pyrethroid-sensing residue,
L1596P, in this linker is also shown in bold.

105

Figure 4-2. Sodium currents from AaNav1-1 and mutant sodium channels. (A) Representative
current traces. The sodium currents were elicited by a 20-ms depolarization to 0 mV from the
holding potential of -120 mV. (B) Sodium current decay. Time constant of sodium current decay
was calculated by fitting the decaying part of current traces, elicited by a series of depolarizing
voltages, with a single exponential function.

106

Figure 4-3. Voltage dependences of activation and inactivation of AaNav1-1 and mutant sodium
channels. (A) & (B) Voltage-dependence of activation. (C) & (D) Voltage-dependence of
inactivation. Data analysis and the protocols for the voltage-dependence of activation and
inactivation are described in Materials and Methods. Symbols represent means, and error bars
indicate SEM values.

107

Figure 4-3 (Cont’d)

108

Figure 4-4. The N1575Y and L1014F double mutations significantly reduced channel sensitivity
to permethrin and deltamethrin. (A) – (D) Tail currents induced by permethrin (1 µM) in
AaNav1-1, N1575Y, L1014F, and N1575+L1014F channels. (E) & (F) Percentage of channel
modification by permethrin and deltamethrin. Percentage of channels modified by pyrethroids
was calculated as described in Materials and Methods. The values of EC20 for permethrin are
0.12, 0.18, 1.0 and 9.8 µM for AaNav1-1, N1575Y, L1014F, and N1575Y+L1014F channels,
respectively. The values of EC20 for deltamethrin are 0.1, 0.11, 1.4, and 5.3 µM for AaNav1-1,
N1575Y, L1014F, and N1575Y+L1014F channels, respectively.

109

Figure 4-4 (Cont’d)

110

Figure 4-5. The N1575Y and L1014S double mutations significantly reduced channel sensitivity
to permethrin and deltamethrin. (A) & (B) Percentage of channel modification by permethrin and
deltamethrin. Percentage of channels modified by pyrethroids was calculated as described in
Materials and Methods. The values of EC20 for permethrin are 0.12, 0.18, 0.8 and 7.0 µM for
AaNav1-1, N1575Y, L1014S, and N1575Y+L1014S channels, respectively. The values of EC20
for deltamethrin are 0.1, 0.11, 0.9, and 3.2 µM for AaNav1-1, N1575Y, L1014S, and
N1575Y+L1014S channels, respectively.

111

Figure 4-6. Effects of the single V1016G mutation and the N1575Y+V1016G double mutations
on AaNav1-1 channel sensitivity to pyethroids. (A) Percentage modification by permethrin. (B)
Percentage modification by deltamethrin. Percentages of channel modification by permethrin (1
µM) or deltamethrin (1 µM) were determined using the method described in Materials and
Methods.
*indicates a significant difference compared to AaNav1-1 using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05).

112

CHAPTER 5
A-TO-I EDITING IN THE DROSOPHILA SODIUM CHANNEL MODIFIES THE GATING
AND SENSITIVITY OF SODIUM CHANNELS TO DELTAMETHRIN AND AV3

113

5.1Abstract
Voltage-gated sodium channels are essential for electrical signaling in the nervous
system. Insects, having only one sodium channel gene, rely on alternative splicing and RNA
editing to generate a large collection of sodium channel variants. So far, 20 A-to-I editing sites
were discovered in the sodium channel transcripts from the adults of Drosophila melanogaster.
However, the functional consequences of these RNA editing events have not been examined.
Our lab members isolated 64 full-length sodium channel cDNA clones from the adult of D.
melanogaster. Functional and pharmacological characterization of these variants in Xenopus
oocytes revealed different sensitivities to a pyrethroid insecticide, deltamethrin, and a site 3
sodium channel toxin, Av3, from the sea anemone Anemonia viridis. One sodium channel
variant, DmNav26, is most resistant to deltamethrin and Av3 but lacks any RNA editing,
suggesting that RNA editing may contribute to the hypersensitivity of other variants to these two
toxins. In this study, I introduced four RNA editing sites and two alternative exons into
DmNav26 to investigate the involvement of RNA editing and alternative splicing in regulating
channel gating properties and sensitivity to deltamethrin and Av3. My analysis identified one
RNA editing event, N1587S, in the linker connecting domains III and IV, caused a 7-mV
hyperpolarizing shift in the voltage-dependence of slow inactivation. Another RNA editing
event, Q1296R, in segment 1 of domain III (IIIS1), enhanced the sensitivity of DmNav26
channels to both deltamethrin and Av3. I also found that replacement of exon k with exon l in
DmNav26 significantly increased persistent sodium current and channel sensitivity to
deltamethrin. My study confirmed and extended the role of alternative splicing and RNA editing
in regulating sodium channel gating as well as alternative splicing in regulating channel
114

sensitivity to pyrethroid insecticides. More significantly, my functional analysis provides the first
direct molecular evidence of the role of RNA editing in regulating the sensitivity of Drosophila
sodium channels to neurotoxins.
5.2. Introduction
Voltage-gated sodium channels are integral transmembrane proteins responsible for the
rapidly rising phase of action potentials in almost all excitable cells (Catterall 2012). Sodium
channel proteins contain four homologous domains (I-IV), each formed by six transmembrane
segments (S1-S6) connected by extracellular and intracellular loops. There are four to seven
positively charged amino acids in the S4 segment in each domain that are important for sodium
channel activation in response to membrane depolarization (Catterall 1986; Guy and
Seetharamulu 1986). S5 and S6 segments of each domain and the membrane-reentrant P-loops
connecting S5 and S6 form the inner part of the channel (Catterall 2000). In response to
depolarization, sodium channels activate and open, and quickly, within a few milliseconds, fast
inactivation occurs and sodium channels close. The linker connecting domains III and IV,
harboring an α helix with an IFMT particle, is essential for sodium channel fast inactivation
(Rohl et al. 1999). In response to prolonged depolarization or repeated stimuli, sodium channels
enter another stage of inactivation called slow inactivation, which requires seconds or even
minutes to recover. This stage is called slow inactivation (Goldin 2003).
In mammals, there are at least nine sodium channel genes, that encode nine different
sodium channel isoforms with different gating properties and different expression patterns in
various cell types, tissues, and developmental states, presumably to fulfill unique physiological
functions in specific neuronal and non-neuronal cells (Yu and Catterall 2003). In insects,
however, there is only one sodium channel gene, such as para in Drosophila melanogaster

115

(Dong 2007). Insects appear to take advantage of extensive alternative splicing and RNA editing
to generate functionally distinct sodium channels (Dong 2007; Olson et al. 2008). Sodium
channel variants from the German cockroach and fruit fly exhibit a broad range in the voltagedependence of activation and inactivation (Lin et al. 2009; Olson et al. 2008; Song et al. 2004).
Alternative splicing has been shown to be responsible for some of the differences in gating
properties (Lin et al. 2009; Tan et al. 2002b). RNA editing is a posttranscriptional process during
which one nucleotide base is altered or converted to another nucleotide, or a nucleotide is deleted
or inserted. Both A-to-I and U-to-C RNA editing have been reported to contribute to functional
diversity of cockroach sodium channels (Liu et al. 2004; Song et al. 2004). 20 A-to-I RNA
editing sites were identified in sodium channel transcripts from different body parts of the adult
D. melanogaster by high throughput sequencing (Fig. 5-1, from Dong lab’s collaborators in Yale
University and Zhejiang University in China). However, the functional consequences of these
RNA editing events have not been examined.
Sodium channels are a target site of many neurotoxins, including pyrethroid insecticides
(Bloomquist 1993; Soderlund and Bloomquist 1989) and polypeptide toxins from the venom of
scorpions and sea anemones, such as Av3 from the sea anemone A. viridis (Moran et al. 2007).
Pyrethroids prefer to bind to open-state sodium channels and inhibit sodium channel inactivation
and deactivation, resulting in prolonged opening of sodium channels (Narahashi 1987). Av3
inhibits sodium channel inactivation similar to the effect of α-scorpion toxins (Moran et al.
2007). Functional and pharmacological characterization of DmNav variants in Xenopus oocytes
showed that the DmNav variants exhibited different sensitivities to both Av3 and a pyrethroid
insecticide, deltamethrin (Olson et al. 2008) ( Fig. 5-2 from Dr. Zhaonong Hu and Fig. 5-3 from
Dr. Rong Gao). Of all sodium channel variants tested, the DmNav26 variant is the most resistant
116

to both deltamethrin and Av3. It contains two alternative exons, a and f, and two mutually
exclusive exons, d and k (Fig. 5-4). Intriguingly, the DmNav26 variant is completely unedited.
In this study, to investigate the role of RNA editing and alternative splicing in regulating
channel gating properties and sensitivity to deltamethrin and Av3, I introduced four RNA editing
sites, K437R, Q1296R, N1300D, and N1587S, into DmNav26, as well as replaced exon k with
exon l and deleted exon f in DmNav26. Substitution of exon k with exon l significantly increased
DmNav26 channel persistent current and channel sensitivity to deltamethrin. I also found that
N1587S substitution caused a hyperpolarizing shift in the voltage-dependence of slow
inactivation. Interestingly, Q1296R substitution increased DmNav26 channel sensitivity to both
deltamethrin and Av3. My results provide the first direct molecular evidence for the role of RNA
editing in regulating Drosophila sodium channel sensitivity to neurotoxins.
5.3. Materials and Methods
5.3.1. Site-directed mutagenesis
Site-directed mutagenesis was performed by PCR using mutant primers and Pfu Turbo
DNA polymerase (Stratagene, La Jolla, CA). All mutagenesis results were confirmed by DNA
sequencing.
5.3.2. Expression of sodium channels in Xenopus oocytes
The procedures for oocyte preparation and cRNA injection are identical to those
described previously (Tan et al. 2005). For robust expression of para sodium channels, cRNA
was coinjected into oocytes with Drosophila melanogaster TipE cRNA (1:1 ratio), which
enhances the expression of insect sodium channels in oocytes.
5.3.3. Electrophysiological recording and analysis
117

The voltage dependence of activation and inactivation was measured using the twoelectrode voltage clamp technique. Methods for two-electrode recording and data analysis were
identical to those described previously (Tan et al. 2005).
The voltage dependence of sodium channel conductance (G) was calculated by
measuring the peak current at test potentials ranging from -80 mV to +65 mV in 5 mV
increments and divided by (V – Vrev), where V is the test potential and Vrev is the reversal
potential for sodium current. Peak conductance values were normalized to the maximal peak
conductance (Gmax) and fitted with a two-state Boltzmann equation of the form G/Gmax = [1 +
-1

exp(V-V1/2)/k] , in which V is the potential of the voltage pulse, V1/2 is the voltage for halfmaximal activation, and k is the slope factor.
The voltage dependence of sodium channel fast inactivation was determined by using 100
ms inactivating prepulses ranging from -120 mV to 10 mV in 5-mV increments from a holding
potential of -120 mV, followed by test pulses to -10 mV for 20 ms. The peak current amplitude
during the test pulse was normalized to the maximum current amplitude and plotted as a function
of the prepulse potential. Data were fitted with a two-state Boltzmann equation of the form
-1

I/Imax = [1 + (exp (V - V1/2)/k)] , in which I is the peak sodium current, Imax is the maximal
current evoked, V is the potential of the voltage prepulse, V1/2 is the half-maximal voltage for
inactivation, and k is the slope factor.
The voltage dependence of sodium channel slow inactivation was measured with 60s
conditioning pulses ranging from -100 mV to 0 mV in 10 mV increments, followed by
repolarization to a holding potential of -120 mV for 100 ms to remove fast inactivation, and at
last a test pulse of -10 mV for 20 ms. The peak current amplitude during the test depolarization
118

was normalized to the maximum current amplitude and plotted against the conditioning pulse
potential. Data were fitted with a two-state Boltzmann equation as above for fast inactivation.
5.3.4. Measurement of tail currents induced by pyrethroids
The method for application of pyrethroids in the recording system was identical to that
described by Tan et al. (Tan et al. 2005). The effects of pyrethrois were measured 10 min after
toxin application. The pyrethroid-induced tail current was recorded during a 100-pulse train of 5
ms step depolarizations from -120 to 0 mV with 5 ms interpulse intervals (Vais 2000). The
percentage of channels modified by pyrethroids was calculated using the equation M = {[Itail/(Eh
– ENa)]/[INa/(Et – ENa)]} X 100 (Tatebayashi and Narahashi 1994), where Itail is the maximal
tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is the
reversal potential for sodium current determined from the current-voltage curve, INa is the
amplitude of the peak current during depolarization before pyrethroids exposure, and Et is the
potential of step depolarization. Dose-response curves were fitted by the Hill equation: M =
n

Mmax/{1 + (Kd/[P]) }, in which [P] represents the concentration of pyrethroids, Kd represents
the concentration of pyrethroid that produced the half-maximal effect, n represents the Hill
coefficient, and Mmax is the maximal percentage of sodium channel modified.
5.3.5. Measurement of sodium channel sensitivity to Av3
The method for Av3 application is the same as that for pyrethroids described above. Peak
sodium current was recorded 10 min after Av3 application. Both the amplitude of the peak
sodium current and the non-inactivating current at the end of the 20 ms depolarization were
measured. Sodium channel sensitivity to Av3 was calculated by normalizing the amplitude of
119

non-inactivating current at 20 ms time point to amplitude of transient peak sodium current and
times 100.
5.3.6. Chemicals
Deltamethrin was kindly provided by Bhupinder Khambay (Rothamsted Research,
Harpenden, UK). Deltamethrin was dissolved in dimethyl sulfoxide (DMSO). The working
concentration was prepared in DN96 recording solution immediately prior to experiments. The
concentration of DMSO in the final solution was <0.5%, which had no effect on the function of
sodium channels.
5.3.7. Statistical analysis
Data were sampled at 50 kHz and filtered at 2 kHz. Leak currents were corrected by p/4
subtraction. pClamp 10.2 software (Axon Instruments Inc., CA) and Origin 8.0 software were
used for data acquisition and analysis. Results are reported as mean ± SEM. Data were analyzed
using one-way analysis of variance (ANOVA), if ANOVA shows a significant effect, Scheffe’s
post hoc comparisons were used to compare the means. Results were considered to be significant
when p < 0.05.
5.4. Results
5.4.1. Substituting exon l for exon k significantly increased the persistent sodium current and
sensitivity of DmNav26 channels to deltamethrin.
The DmNav26 variant contains two alternative exons, a and f, and two mutually
exclusive exons, d and k. Based on deltamethrin screening results and alternative exon usage, I
found that most of the variants that have alternative exon f are resistant to deltamethrin. We also
found that only two variants have exon k and both of them are resistant to deltamethrin. Because

120

of these findings, two mutant sodium channels were created in DmNav26: one mutant without
-

+

exon f (exon f ), and the other with exon l (exon l ).
Removing exon f or replacing exon k with exon l did not alter DmNav26 channel
voltage-dependence of activation, fast inactivation, or slow inactivation (Table 5-1; Fig. 5-5A
and B). Replacement of exon k with exon l significantly increased the persistent sodium current
(Fig. 5-5C and D). Removal of exon f did not alter the persistent sodium current (Fig. 5-5D).
Replacement of exon k with exon l significantly increased the sensitivity of DmNav26
channels to deltamethrin (Fig. 5-6C), as evident in the larger amplitude of tail current induced by
1 µM deltamethrin (Fig. 5-6B). Pyrethroid-induced tail current decay was calculated by fitting
+

the tail current with a double-exponential function. Exon l mutant sodium channel had similar
tail current decay with that of DmNav26 (Table 5-2).
Av3 inhibits sodium channel inactivation (Fig. 5-11B). Sodium channel sensitivity to
Av3 was calculated by normalizing the non-inactivating current at the end of 20 ms
depolarization to peak current as indicated in Fig. 5-11B. About 19% of current from DmNav26
was modified by application of 250 nM Av3 (Fig. 5-10C). Removal of exon f or replacement of
exon k with exon l did not alter the sensitivity of DmNav26 channels to Av3 (Fig. 5-11C).
5.4.2. Three amino acid changes unique to DmNav26 did not confer channel resistance to
deltamethrin and Av3.
Although DmNav26 is not RNA-edited, it contains three unique amino acid changes
(E299Q, L1363F, and P1977S) that are not found in other DmNav variants. These changes could
121

be due to nucleotide polymorphisms or errors introduced during real-time PCR cloning. To
determine whether any of these changes are responsible for the high level of resistance to
deltamethrin and Av3, I replaced these residues with those in the other DmNav variants. For
example, for the E299Q change in DmNav26, E299 is found in other variants. Therefore, I
replaced Q299 with E299 using site-directed mutagenesis. First, I examined the gating properties
of Q299E, F1363L, and S1977P mutant channels. None of them altered the voltage-dependence
of activation or fast inactivation (Table 5-1; Fig. 5-7B and C). Then I examined the sensitivity of
the mutant channels to deltamethrin, a type II pyrethroid that requires sodium channels to be in
open state for its action. I used a multiple pre-pulse protocol to test the deltamethrin-induced tail
current (Fig 5-8B). The percentage of modified channels was quantified using the method
developed by Tatebayashi and Narahashi 1994 (Tatebayashi and Narahashi 1994). None of three
amino acid changes altered DmNav26 channel sensitivity to deltamethrin or Av3 (Fig. 5-8C).
5.4.3. N1587S in the linker connecting domains III and IV caused a hyperpolarizing shift in the
voltage-dependence of slow inactivation.
The fact that variants that are extensively RNA-edited are more sensitive to deltamethrin
than DmNav26 suggests that RNA editing enhances the sensitivity of these variants to
deltamethrin. To test this hypothesis, four A-to-I editing sites (K437R, Q1296R, N1300D, and
N1587S) were chosen based on the correlation between the level of deltamethrin sensitivity and
the presence or absence of RNA editing sites. The four editing sites were individually introduced
into DmNav26 and their gating properties were examined.
None of these four RNA editing events alter the voltage-dependence of activation and
fast inactivation (Table 5-1; Fig. 5-9B and C). However, the N1587S substitution in the linker
122

connecting domains III and IV caused a hyperpolarizing shift in DmNav26 voltage-dependence
of slow inactivation (Table 5-1; Fig. 5-9D).
5.4.4. Q1296R substitution increased DmNav26 channel sensitivity to deltamethrin and Av3.
I then examined the sensitivity of mutant channels to deltamethrin and Av3. The
pyrethroid-induced tail current was recorded during a 100-pulse train of 5-ms step
depolarizations from – 120 to 0 mV with 5-ms interpulse intervals (Vais et al., 2000). The
percentage of sodium channel modification by pyrethroids was calculated as described in
Materials and Methods. The Q1296R substitution, located in the linker connecting domains II
and III, significantly increased the amplitude of tail current induced by deltamethrin (Fig. 510B), and thereby the sensitivity to deltamethrin (Fig. 5-10C). Interestingly, the deltamethrin
induced tail current decayed much slowly for Q1296R mutant channels than for DmNav26
channels (Table 5-2). Slower decay of tail means slower close of sodium channels and stronger
activity of pyrethroids. It also indicates that sodium channels are more sensitive to pyrethroids.
None of other three mutants altered DmNav26 channel percentage modification or tail current
decay (Fig. 5-10C).
Q1296R substitution also significantly increased DmNav26 channel sensitivity to Av3
(Fig. 5-11C). The percentage of the modified current was increased from about 19% to 40%.
None of other mutants altered DmNav26 sensitivity to Av3 (Fig. 5-11C).
5.5. Discussion
In this study, my functional analysis showed that: 1) RNA editing at the Q1296R site in
IIIS1 significantly increased the sensitivity of DmNav26 channels to deltamethrin; 2) the same
123

RNA editing also increased the channel sensitivity to Av3; 3) RNA editing at the N1587S site in
the linker connecting domains III and IV caused a hyperpolarizing shift in the voltagedependence of slow inactivation; and 4) the mutually exclusive exons k and l were involved in
regulating the channel sensitivity to deltamethrin, i.e., replacement of exon k with exon l
significantly increased the sensitivity of DmNav26 channels to deltamethrin.
Alternative splicing has been shown to generate functionally distinct sodium channels
(Du et al. 2009; Lin et al. 2009; Olson et al. 2008). Both naturally occurring alternative splice
variants from different parts of adults of D. melanogaster and constructed exon-specific DmNav
variants based on sodium channel variants from larval D. melanogaster exhibited diverse gating
properties (Lin et al. 2009; Olson et al. 2008). Mutually exclusive exons k and l were shown to
regulate persistent sodium currents in a constructed exon-specific variant: substitution of exon k
with exon l significantly increased the persistent sodium current (Lin et al. 2009). My results
confirmed this role of exons k and l in a naturally occurring variant, DmNav26.
Several RNA editing sites in the cockroach sodium channels have previously been shown
to alter sodium channel gating properties in Xenopus oocytes. For example, an U-to-C RNA
editing resulting in a phenylalanine to serine mutation in the C-terminus of a cockroach sodium
channel at position 1919 induced a persistent sodium current (Liu et al. 2004). In addition, two
RNA editing events (one U-to-C and one A-to-I) were confirmed to alter the voltage-dependent
gating properties of cockroach sodium channels (Song et al. 2004). Interestingly, these RNA
editing events occurred in a tissue-specific and development-specific manner (Song et al. 2004),
indicating their potential unique roles in vivo. Here, my results provide another example of
gating modification by an A-to-I editing. In this case, RNA editing affect slow inactivation

124

which is critical for regulating membrane excitability, action potential patterns and spike
frequency adaptation (Vilin and Ruben 2001). Collectively, these results show that RNA editing
can cause various modifications of channel gating, which likely respond to the physiological
needs of neurons where these RNA editing events occur.
The role of alternative splicing in regulating sodium channel sensitivity to pyrethroids has
been documented in the cockroach sodium channels. Deletion of residue G1111 as a result of
alternative splicing in BgNav1-1 of a cockroach sodium channel increased channel sensitivity to
pyrethroids (Du et al. 2009). Also, a pair of mutually exclusive exons, G1 and G2, were
confirmed to be involved in channel sensitivity to pyrethroids: inclusion of exon G2 made
sodium channels more resistant to pyrethroids (Tan et al. 2002b). In D. melanogaster, mutually
exclusive exons k and l are homologous to G2 and G1 in the cockroach sodium channel. My
results confirmed that like exon G2, exon k contributes to sodium channel resistance to
pyrethroids. Exon k or l is located at the IIIS3-S4 region. It is highly possible that substitution of
exon k with exon l modified movement of IIIS4, which in turn altered movement of IIIS6 (part
of pyrethroid receptor 1 as introduced below) and thus altered sodium channel sensitivity to
pyrethroids.
Prior to this study, the role of RNA editing in regulating channel sensitivity to pyrethroid
insecticides was unknown. Glycine substitution of a negatively charged amino acid at the
position 802 in a cockroach sodium channel variant significantly reduced the sensitivity of the
variant to deltamethrin (Du et al. 2010). However, whether this substitution is the result of RNA
editing has not been determined (Du et al. 2010). An RNA editing mutation I951V was found by
comparing the cDNA and genomic DNA in pyrethroid-resistant Helicoverpa zea populations
(Hopkins and Piertrantonio 2010). Whether this RNA mutation contributes to sodium channel
125

resistance to pyrethroids is unknown. My study provides the first experimental evidence
supporting the role of RNA editing in regulating the sensitivity of sodium channels to a
pyrethroid and also a venom toxin.
How the Q1296R substitution enhances channel sensitivity to deltamethrin and Av3
remains to be determined. It could directly or indirectly reduce toxin binding. Two pyrethroid
receptors have been reported based on computer modeling and electrophysiological analyses (Du
et al. 2013; O'Reilly et al. 2006). The first pyrethroid receptor was proposed in the open state of
the house fly sodium channel based on the crystal structure of voltage-gated potassium channel
(O'Reilly et al. 2006). This model indicated that pyrethroids bind to a triangle pocket composed
of the linker connecting S4 and S5 in domain II, domain II S5, and domain III S6. The second
pyrethroid receptor was built on a mosquito sodium channel based on the crystal structure of a
voltage-gated potassium channel and a bacterial sodium channel (Du et al. 2013). This second
pyrethroid receptor was composed of the linker connecting S4 and S5 in domain I, domain I S5,
and domain II S6 (Du et al. 2013). Av3 is proposed to bind to the extracellular loops connecting
S1-S2 and S3-S4 in domain IV and the loop of SS2-S6 in domain one (Wang et al. 2011). This
Q1296R substitution is not located or close to either of the pyrethroid binding sites or Av3
receptor site and therefore unlikely that this substitution directly interfere with toxin binding.
Although pyrethroids and Av3 are two distinct toxins, they are not unrelated. First, both
toxins have similarity regarding the effects on sodium channels: pyrethroids prefer to bind to the
open state of sodium channels and inhibit inactivation and deactivation (Soderlund 2012), while
Av3 inhibits sodium channel inactivation (Moran et al., 2007). Second, evidence has shown that
both toxins exhibit synergistic binding effects (Gilles et al., 2003). Several amino acid changes
were predicted to alter channel sensitivity to pyrethroids by modulating sodium channel

126

activation and deactivation kinetics, which could counteract the effects of pyrethroids (Du et al.
2010; Oliveira et al. 2013). It is highly possible that Q1296R substitution allosterically altered
DmNav26 channel gating properties and subsequently enhanced the effects of both toxins.
In conclusion, my study confirmed the effects of alternative splicing and RNA editing on
modulating sodium channel gating properties in Xenopus oocytes, indicating that both play
significant roles in regulating insect neuronal excitability in vivo. The results of this study
verified that mutually exclusive exons k and l are involved in channel sensitivity to pyrethroid
insecticides. My study also provided the first molecular evidence that RNA editing is involved in
modulating sodium channel sensitivity to neurotoxins.

127

Table 5-1. Voltage-dependence of activation, fast inactivation, and slow inactivation of
DmNav26 and its mutants.
n
Activation
Fast Inactivation
Slow inactivation
V1/2 (mV) k
DmNav26 -24.7 ± 0.6 3.4 ± 0.6

V1/2 (mV) k

V1/2 (mV) k

-40.8 ± 0.4 4.2 ± 0.2

-45.6 ± 0.2 5.0 ± 0.3 18

+

-28.2 ± 1.9 4.5 ± 1.0

-40.0 ± 0.4 4.2 ± 0.1

-42.9 ± 0.3 5.1 ± 0.2 12

Exon f

-

-26.5 ± 2.7 3.4 ± 0.8

-41.6 ± 0.5 4.3 ± 0.1

-43.3 ± 0.1 5.5 ± 0.4 12

Q299E

-25.6 ± 0.9 3.5 ± 0.6

-40.2 ± 0.5 4.0 ± 0.1

-46.2 ± 0.3 5.1 ± 0.2 10

K437R

-26.2 ± 1.2 3.9 ± 0.5

-43.8 ± 0.7 4.1 ± 0.7

-41.2 ± 1.0 5.7 ± 0.5 13

Q1296R

-28.1 ± 1.3 3.5 ± 0.2

-42.6 ± 0.3 4.1 ± 0.1

-48.3 ± 1.7 4.6 ± 0.1 13

N1300D

-26.7 ± 1.6 3.7 ± 0.5

-44.7 ± 2.9 4.4 ± 0.3

-44.6 ± 0.5 5.2 ± 0.2 12

F1363L

-27.6 ± 1.5 3.3 ± 0.4

-43.2 ± 0.7 4.2 ± 0.1

-40.9 ± 0.3 5.1 ± 0.2 11

N1587S

-26.1 ± 2.3 3.9 ± 1.1

-41.0 ± 0.5 3.9 ± 0.1

-52.9 ± 0.3* 4.5 ± 0.1 10

S1977P

-27.2 ± 1.3 3.2 ± 0.6

-42.6 ± 0.8 4.0 ± 0.2

-42.7 ± 0.6 5.1 ± 0.4 6

Exon l

The voltage-dependence of activation, fast inactivation, and slow inactivation were fitted with
two-state Boltzmann equations, as described in materials and methods, to determine V1/2, the
voltage for half-maximal conductance or inactivation and k, the slope for conductance or
inactivation. Each value represents the mean ± SEM.
* Significant difference from that of DmNav26 channel using one-way ANOVA with Scheffe’s
post hoc analysis (p < 0.05).

128

Table 5-2. Time constant of tail current decay induced by deltamethrin.
Na+ channel type

τ1 (s)

τ2 (s)

n

DmNav26

1.16 ± 0.17

0.20 ± 0.03

12

Q1296R

1.83 ± 0.25*

0.20 ± 0.02

11

+

1.47 ± 0.18

0.24 ± 0.03

9

exon l

Tail current induced by 1 µM deltamethrin was fitted with double exponential equation. Each
value represents the mean ± SEM.
n: number of oocytes.
* Significant difference compared with DmNav26 channel using one-way ANOVA with
Scheffe’s post hoc analysis (p < 0.05).

129

Figure 5-1. Localization of 20 A-to-I RNA editing sites in the Drosophila sodium channel. From
our collaborators in Zhejiang University in China and Yale University.

130

Figure 5-2. Differential sensitivity of DmNav sodium channel variants to deltamethrin. Sodium
channel variants were transiently expressed individually in Xenopus oocytes. Percentages of
channels modified by 1 µM pyrethroids was calculated using the equation
M={[Itail/(Eh−ENa)]/[INa/(Et−ENa)]}×100 (Tatebayashi and Narahashi, 1994),where Itail is the
maximal tail current amplitude, Eh is the potential to which the membrane is repolarized, ENa is
the reversal potential for sodium current determined from the current–voltage curve, INa is the
amplitude of the peak current during depolarization before pyrethroid exposure, and Et is the
potential of step depolarization. (Results from Dr. Zhaonong Hu).

131

Figure 5-3. Differential sensitivity of DmNav sodium channel variants to Av3. Sodium channel
variants were transiently expressed individually in Xenopus oocytes. Sodium channel sensitivity
to 250 nM of Av3 was measured by normalizing peak of non-inactivating current at end of 20 ms
depolarization to peak of transient sodium current. (Results from Dr. Rong Gao).

132

Figure 5-4. Topology of the sodium channel variant DmNav26. Four RNA editing sites
discovered in other DmNav sodium channels were shown in black and three non-RNA editing
amino acid changes were in gray.DmNav26 contains two optional exons, a and f, and two
mutually exclusive exons, d and k.

133

Figure 5-5. Replacement of exon k with exon l significantly increased the sensitivity of
DmNav26 channel to deltamethrin. (A) Localization of the mutually exclusive exons k/l and the
alternative exon f. (B) Representative tail currents induced by 1 µM deltamethrin. (C) Percentage
of channel modification by deltamethrin. Sodium channel sensitivity to deltamethrin was tested
and analyzed as described in Materials and Methods.
* Significant difference compared with DmNav26 using one-way ANOVA with Scheffe’s post
hoc analysis (p < 0.05).

134

Figure 5-5. (Cont’d)

135

Figure 5-6. Replacement of exon k with exon l increased the persistent sodium current. (A)
Voltage-dependence of activation. (B) Voltage-dependence of fast inactivation. (C)
+
Representative current traces of DmNav26 and exon l mutant channels induced by a step
depolarization from a holding potential of -120 mV to 0 mV. (D) Percentage of persistent current
+
of DmNav26, exon f , and exon l channels. Percentage of persistent current was normalized to
the amplitude of peak sodium current.
* Significant difference compared with DmNav26 using one-way ANOVA with Scheffe’s post
hoc analysis (p < 0.05).

136

Figure 5-6. (Cont’d)

137

Figure 5-7. Voltage-dependence of activation and fast inactivation of DmNav26 and Q299E,
F1363L, and S1977P mutants. (A) Position of all three non-RNA editing amino acid changes.
(B) Voltage-dependence of activation. (C) Voltage-dependence of fast inactivation. Voltagedependence of activation and fast inactivation were tested and analyzed as described in Materials
and Methods.

138

Figure 5-7. (Cont’d)

139

Figure 5-8. DmNav26 and Q299E, F1363L, and S1977P mutant channels have similar sensitivity
to deltamethrin. (A) Topological localization of all three none RNA editing amino acid changes.
(B) Tail current of DmNav26 induced by 1 µM deltamethrin and the formula of calculating
sodium channel percentage modification by pyrethroids. (C) Percentage modified channels of
DmNav26 and three none RNA editing mutant channels. Sodium channel sensitivity to
deltamethrin was tested and analyzed as described in Materials and Methods.

140

Figure 5-8. (Cont’d)

141

Figure 5-9. Voltage-dependence of activation, fast inactivation, and slow inactivation of
DmNav26 and K437R, Q1296R, N1300D, and N1587S mutants. (A) Topological localization of
all four RNA editing events in DmNav26. (B) Voltage-dependence of activation. (C) Voltagedependence of fast inactivation. (D) Voltage-dependence of slow inactivation. Sodium channel
voltage-dependence of activation, fast inactivation and slow inactivation were tested and
analyzed as described in Materials and Methods.

142

Figure 5-9. (Cont’d)

143

Figure 5-10. Q1296R substitution increased DmNav26 sensitivity to deltamethrin. (A)
Topological localization of the Q1296R mutation. (B) Representative tail currents of DmNav26
and Q1296R mutant channels induced by 1 µM deltamethrin. (C) Percentage modified channels
of DmNav26 and all four RNA editing event mutant channels. Sodium channel sensitivity to
deltamethrin was tested and analyzed as described in Materials and Methods.
* Significant difference compared with DmNav26 using one-way ANOVA with Scheffe’s post
hoc analysis (p < 0.05).

144

Figure 5-10. (Cont’d)

145

Figure 5-11. Q1296R mutation increased DmNav26 sensitivity to Av3. (A) Topological
localization of Q1296R mutation. (B) Representative sodium current traces before and after Av3
application. Dashed line indicates the time point where sodium channel inactivation was
measured. (C) Percentage modification of DmNav26 and mutant channels by 250 nM Av3.
Percentage modification was calculated by normalizing the inactivation current at 20 ms point to
the peak sodium current.
* Significant difference compared with DmNav26 using one-way ANOVA with Scheffe’s post
hoc analysis (p < 0.05).

146

Figure 5-11. (Cont’d)

147

CHAPTER 6
FUTURE DIRECTIOIN

148

Results of this dissertation show that insect sodium channel auxiliary subunits and RNA
editing events have diverse effects on the gating and pharmacological properties of sodium
channels expressed in Xenopus oocytes. These findings indicate that auxiliary subunits and RNA
editing events play important roles in regulating neuronal excitability in vivo. However, how
these auxiliary subunits and RNA editing events regulate neuronal activity in vivo is still
unknown.
In order to understand the physiological roles of sodium channel auxiliary subunits and
RNA editing events, transgenic insects should be generated first and then both behavioral assays
and electrophysiological experiments will be necessary to compare the mutant insects with the
wild-type insects. With the well-developed genetic tools, D. melanogaster would be the ideal
model insect to investigate the function of sodium channel auxiliary subunits and RNA editing
events. A few genetic and molecular methods could be used to make transgenic flies. For
auxiliary subunits, we could knockout TEH genes by homologous recombination method. We
could also reduce gene expression by RNAi technique. For RNA editing events, we could
introduce RNA editing into the whole body of Drosophila by homologous recombination
method. This will help broadly understand the role of RNA editing. Or we could use Gal4-UAS
technique to introduce RNA editing event into certain neuronal circuits or certain type of
neurons, which will help precisely explore the role of RNA editing. For flies that having
confirmed RNA editing events, we could study the physiological role of these RNA editing
events by removing them one by one with homologous recombination technique. Extensive
experiments including behavioral assays and electrophysiological recordings will be required to
study both mutant flies and wild-type flies. Insect behavioral assays include: heat shock assay,
climbing assay, bang-sensitivity assay, and insecticides bioassay. More extensive in vitro

149

electrophysiological recording in dissociated neurons or in vivo recording of specific neuronal
circuits should be used to measure the neuronal activities. Results of both behavioral assays and
neuronal activities of mutant flies will be compared with that of wild-type flies to identify the
physiological roles of sodium channel auxiliary subunits or RNA editing events.
In addition, questions regarding insect sodium channel composition, tissue distribution of
insect sodium channel auxiliary subunits, and the molecular mechanism of TipE or TEH1-4modulated sodium channel gating will also need to be addressed.
To clarify the composition of insect sodium channels, we could do protein-protein
interaction experiments, like protein complex immunoprecipitation (Co-IP). RNA in situ
hybridization or immunohistochemistry experiments could be used to investigate the expression
pattern of TipE and TEH1-4 in different cell types, tissues, or developmental stages. To
understand the molecular basis of how TipE or THE1-4 proteins regulate sodium channel gating
properties, we could generate chimeras with different auxiliary subunits. Site-directed
mutagenesis will also be used to understand the molecular basis of interaction between sodium
channel and auxiliary subunits.
In our isolated DmNav sodium channel variants, many amino acid changes have not been
confirmed RNA editing events yet. More work will be required to confirm whether they are
RNA editing events and their functions in regulating gating and pharmacological properties of
sodium channels expressed in Xenopus oocytes.
More work or more defined and advanced high throughput sequencing method will be
needed to confirm low frequency RNA editing events in insects. It will also be interesting to
investigate the function of the rest of confirmed RNA editing events in regulating the gating and
pharmacological properties of DmNav variants expressed in Xenopus oocytes.
150

BIBLIOGRAPHY

151

BIBLIOGRAPHY

Agnew, WS, Moore, AC, Levinson, SR and Raftery, MA (1980) Identification of a large
molecular weight peptide associated with a tetrodotoxin binding protein from the
electroplax of Electrophorus electricus. Biochem Biophys Res Commun 92: 860-6.
Alzheimer, C, Schwindt, PC and Crill, WE (1993) Modal gating of Na+ channels as a
mechanism of persistent Na+ current in pyramidal neurons from rat and cat sensorimotor
cortex. J Neurosci 13: 660-73.
Aman, TK, Grieco-Calub, TM, Chen, C, Rusconi, R, Slat, EA, Isom, LL and Raman, IM (2009)
Regulation of persistent Na current by interactions between beta subunits of voltagegated Na channels. J Neurosci 29: 2027-42.
Beneski, DA and Catterall, WA (1980) Covalent labeling of protein components of the sodium
channel with a photoactivable derivative of scorpion toxin. Proc Natl Acad Sci 77: 63943.
Bezanilla, F and Armstrong, CM (1977) Inactivation of the sodium channel. I. Sodium current
experiments. J Gen Physiol 70: 549-66.
Bloomquist, JR (1993) Toxicology, mode of action and target site-mediated resistance to
insecticides acting on chloride channels. Comp Biochem Physiol 106C: 301-314.
Bloomquist, JR and Soderlund, DM (1988) Pyrethroid insecticides and DDT modify alkaloiddependent sodium channel activation and its enhancement by sea anemone toxin. Mol
Pharmacol 33: 543-550.
Borjesson, SI and Elinder, F (2008) Structure, function, and modification of the voltage sensor in
voltage-gated ion channels. Cell Biochem Biophys 52: 149-74.
Brackenbury, WJ and Isom, LL (2011) Na Channel beta Subunits: Overachievers of the Ion
Channel Family. Front Pharmacol 2: 53.
Brengues, C, Hawkes, NJ, Chandre, F, McCarroll, L, Duchon, S, Guillet, P, Manguin, S,
Morgan, JC and Hemingway, J (2003) Pyrethroid and DDT cross-resistance in Aedes
aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med
Vet Entomol 17: 87-94.
Brown, GB, Gaupp, JE and Olsen, RW (1988) Pyrethroid insecticides: stereospecific allosteric
interaction with the batrachotoxinin-A benzoate binding site of mammalian voltagesensitive sodium channels. Mol Pharmacol 34: 54-59.

152

Brun-Barale, A, Bouvier, JC, Pauron, D, Berge, JB and Sauphanor, B (2005) Involvement of a
sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and
development of a diagnostic test. Pest Manag Sci 61: 549-54.
Burton, MJ, Mellor, IR, Duce, IR, Davies, TGE, Field, LM and Williamson, MS (2011)
Differential resistance of insect sodium channels with kdr mutations to deltamethrin,
permethrin and DDT. Insect Biochem Mol Biol 41: 723-732.
Byerly, L and Leung, HT (1988) Ionic currents of Drosophila neurons in embryonic cultures. J
Neurosci 8: 4379-93.
Casida, JE (1980) Pyrethrum flowers and pyrethroid insecticides. Environ Health Perspect 34:
189-202.
Catterall, W and Epstein, PN (1992) Ion channels. Diabetologia 35 Suppl 2: S23-33.
Catterall, WA (1986) Molecular properties of voltage-sensitive sodium channels. Annu Rev
Biochem 55: 953-85.
Catterall, WA (2000) From ionic currents to molecular mechanisms: the structure and function
of voltage-gated sodium channels. Neuron 26: 13-25.
Catterall, WA (2002) Molecular mechanisms of gating and drug block of sodium channels.
Novartis Found Symp 241: 206-18.
Catterall, WA (2012) Voltage-gated sodium channels at 60: structure, function and
pathophysiology. J Physiol 590: 2577-89.
Chahine, M, Chatelier, A, Babich, O and Krupp, JJ (2008) Voltage-gated sodium channels in
neurological disorders. CNS Neurol Disord Drug Targets. 7: 144-58.
Chahine, M, Ziane, R, Vijayaragavan, K and Okamura, Y (2005) Regulation of Nav channels in
sensory neurons. Trends Pharmacol Sci 26: 496-502.
Chen, C and Cannon, SC (1995) Modulation of Na+ channel inactivation by the beta 1 subunit: a
deletion analysis. Pflugers Arch 431: 186-95.
Chen, H, Githeko, AK, Githure, JI, Mutunga, J, Zhou, G and Yan, G (2008a) Monooxygenase
levels and knockdown resistance (kdr) allele frequencies in Anopheles gambiae and
Anopheles arabiensis in Kenya. J Med Entomol 45: 242-250.
Chen, Y, Yu, FH, Sharp, EM, Beacham, D, Scheuer, T and Catterall, WA (2008b) Functional
properties and differential neuromodulation of Na(v)1.6 channels. Mol Cell Neurosci 38:
607-15.
Clay, JR (2003) On the persistent sodium current in squid giant axons. J Neurophysiol 89: 640-4.
153

Crill, WE (1996) Persistent sodium current in mammalian central neurons. Annu Rev Physiol 58:
349-62.
Cusdin, FS, Nietlispach, D, Maman, J, Dale, TJ, Powell, AJ, Clare, JJ and Jackson, AP (2010)
The sodium channel beta3-subunit induces multiphasic gating in Nav1.3 and affects fast
inactivation via distinct intracellular regions. J Biol Chem 285: 33404-12.
Defaix, A and Lapied, B (2005) Role of a novel maintained low-voltage-activated inward current
permeable to sodium and calcium in pacemaking of insect neurosecretory neurons. Invert
Neurosci 5: 135-46.
Derst, C, Walther, C, Veh, RW, Wicher, D and Heinemann, SH (2006) Four novel sequences in
Drosophila melanogaster homologous to the auxiliary Para sodium channel subunit TipE.
Biochem Biophys Res Commun 339: 939-48.
Dong, K (1997) A single amino acid change in the para sodium channel protein is associated
with knockdown-resistance kdr to pyrethroid insecticides in German cockroach. Insect
biochemistry and molecular biology 27: 93-100.
Dong, K (2007) Insect sodium channels and insecticide resistance. Invert Neurosci 7: 17-30.
Dong, K (2010) Progress in insect sodium channel research. In: Insect Pharmacology, Channels,
Receptors, Toxins and Enzymes (Gilbert, LI and Gill, SS, eds.). pp. 25-27. Academic
Press.
Dong, K and Scott, JG (1994) Linkage of kdr-type resistance and the para-homologous sodium
channel in German cockroaches (Blattella germanica). Insect Biochem Mol Biol 24: 647654.
Drali, R, Benkouiten, S, Badiaga, S, Bitam, I, Rolain, JM and Brouqui, P (2012) Detection of a
knockdown resistance mutation associated with permethrin resistance in the body louse
Pediculus humanus corporis by use of melting curve analysis genotyping. J Clin
Microbiol 50: 2229-33.
Du, Y, Nomura, Y, Luo, N, Liu, Z, Lee, JE, Khambay, B and Dong, K (2009) Molecular
determinants on the insect sodium channel for the specific action of type II pyrethroid
insecticides. Toxicol Appl Pharmacol 234: 266-72.
Du, Y, Nomura, Y, Satar, G, Hu, Z, Nauen, R, He, SY, Zhorov, BS and Dong, K (2013)
Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel.
Proc Natl Acad Sci.
Du, Y, Song, W, Groome, JR, Nomura, Y, Luo, N and Dong, K (2010) A negative charge in
transmembrane segment 1 of domain II of cockroach sodium channel is critical for
channel gating and action of pyrethroid insecticides. Tox Appl Pharmacol 247: 53-59.
154

Elliott, M (1980) Established pyrethroid insecticides. Pestic Sci 11: 119-128.
Elliott, M, Farnham, AW, Janes, NF, Needham, PH, Pulman, DA and Stevenson, JH (1973) A
photostable pyrethroid. Nature 246: 169-70.
Enayati, AA, Vatandoost, H, Ladonni, H, Townson, H and Hemingway, J (2003) Molecular
evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito
Anopheles stephensi. Med Vet Entomol 17.
Enomoto, A, Han, JM, Hsiao, CF, Wu, N and Chandler, SH (2006) Participation of sodium
currents in burst generation and control of membrane excitability in mesencephalic
trigeminal neurons. J Neurosci 26: 3412-22.
Fahmi, AI, Patel, M, Stevens, EB, Fowden, AL, John, JE, 3rd, Lee, K, Pinnock, R, Morgan, K,
Jackson, AP and Vandenberg, JI (2001) The sodium channel beta-subunit SCN3b
modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart. J
Physiol 537: 693-700.
Feng, G, Deak, P, Chopra, M and Hall, LM (1995) Cloning and functional analysis of TipE, a
novel membrane protein that enhances Drosophila para sodium channel function. Cell 82:
1001-11.
Fleidervish, IA and Gutnick, MJ (1996) Kinetics of slow inactivation of persistent sodium
current in layer V neurons of mouse neocortical slices. J Neurophysiol 76: 2125-30.
Fletcher, EV, Kullmann, DM and Schorge, S (2011) Alternative splicing modulates inactivation
of type 1 voltage-gated sodium channels by toggling an amino acid in the first S3-S4
linker. J Biol Chem 286: 36700-8.
Gammon, DW, Brown, MA and Casida, JE (1981) Two classes of pyrethroid action in the
cockroach. Pestic Biochem Physiol 15: 181-191.
Gastaldi, M, Robaglia-Schlupp, A, Massacrier, A, Planells, R and Cau, P (1998) mRNA coding
for voltage-gated sodium channel beta2 subunit in rat central nervous system: cellular
distribution and changes following kainate-induced seizures. Neurosci Lett 249: 53-6.
Ginsburg, K and Narahashi, T (1999) Time course and temperature dependence of allethrin
modulation of sodium channels in rat dorsal root ganglion cells. Brain Res 847: 38-49.
Goldin, AL (1992) Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207:
266-79.
Goldin, AL (2003) Mechanisms of sodium channel inactivation. Curr Opin Neurobiol 13: 28490.

155

Goldin, AL, Barchi, RL, Caldwell, JH, Hofmann, F, Howe, JR, Hunter, JC, Kallen, RG, Mandel,
G, Meisler, MH, Netter, YB, Noda, M, Tamkun, MM, Waxman, SG, Wood, JN and
Catterall, WA (2000) Nomenclature of voltage-gated sodium channels. Neuron 28: 3658.
Greenwood, IA and Large, WA (1995) Comparison of the effects of fenamates on Ca-activated
chloride and potassium currents in rabbit portal vein smooth muscle cells. Br J
Pharmacol. 116: 2939-48.
Grolleau, F and Lapied, B (2000) Dorsal unpaired median neurones in the insect central nervous
system: towards a better understanding of the ionic mechanisms underlying spontaneous
electrical activity. J Exp Biol 203: 1633-48.
Guy, HR and Seetharamulu, P (1986) Molecular model of the action potential sodium channel.
Proc Natl Acad Sci 83: 508-12.
Hall, CA (1978) The efficiency of cypermethrin (NRDC 149) for the treatment and eradication
of the sheep louse Damalinia ovis. Aust Vet J 54: 471-2.
Hanrahan, CJ, Palladino, MJ, Ganetzky, B and Reenan, RA (2000) RNA editing of the
Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental
regulation. Genetics 155: 1149-60.
Hartshorne, RP and Catterall, WA (1981) Purification of the saxitoxin receptor of the sodium
channel from rat brain. Proc Natl Acad Sci 78: 4620-4.
Hartshorne, RP, Messner, DJ, Coppersmith, JC and Catterall, WA (1982) The saxitoxin receptor
of the sodium channel from rat brain. Evidence for two nonidentical beta subunits. J Biol
Chem 257: 13888-91.
He, L, Li, T, Zhang, L and Liu, N (2012) Multiple sodium channel variants in the mosquito
Culex quinquefasciatus. Int J Biol Sci 8: 1291-309.
Henry-Halldin, CN, Nadesakumaran, K, Keven, JB, Zimmerman, AM, Siba, P, Mueller, I,
Hetzel, MW, Kazura, JW, Thomsen, E, Reimer, LJ and Zimmerman, PA (2012)
Multiplex assay for species identification and monitoring of insecticide resistance in
Anopheles punctulatus group populations of Papua New Guinea. Am J Trop Med Hyg 86:
140-51.
Hodges, DD, Lee, D, Preston, CF, Boswell, K, Hall, LM and O'Dowd, DK (2002) tipE regulates
Na+-dependent repetitive firing in Drosophila neurons. Mol Cell Neurosci. 19: 402-16.
Hodgkin, AL and Huxley, AF (1952) A quantitative description of membrane current and its
application to conduction and excitation in nerve. J Physiol 117: 500-44.

156

Hogg, RC, Wang, Q and Large, WA (1994) Action of niflumic acid on evoked and spontaneous
calcium-activated chloride and potassium currents in smooth muscle cells from rabbit
portal vein. Br J Pharmacol. 112: 977-84.
Hopkins, BW and Piertrantonio, PV (2010) The Helicoverpa zea (Boddie) (Lepidoptera:
Noctuidae) voltage-gated sodium channel and mutations associated with pyrethroid
resistance in field-collected adult males. Insect Biochem. Molec. Biol. 40: 385-393.
Hotson, JR, Prince, DA and Schwartzkroin, PA (1979) Anomalous inward rectification in
hippocampal neurons. J Neurophysiol 42: 889-95.
Ingles, PJ, Adams, PM, Knipple, DC and Soderlund, DM (1996) Characterization of voltagesensitive sodium channel gene coding sequences from insecticide-susceptible and
knockdown-resistant house fly strains. Insect Biochem Mol Biol 26: 319-326.
Isom, LL (2002a) Beta subunits: players in neuronal hyperexcitability? Novartis Found Symp
241: 124-38; discussion 138-43, 226-32.
Isom, LL (2002b) The role of sodium channels in cell adhesion. Front Biosci 7: 12-23.
Isom, LL, De Jongh, KS, Patton, DE, Reber, BF, Offord, J, Charbonneau, H, Walsh, K, Goldin,
AL and Catterall, WA (1992) Primary structure and functional expression of the beta 1
subunit of the rat brain sodium channel. Science 256: 839-42.
Isom, LL, Ragsdale, DS, De Jongh, KS, Westenbroek, RE, Reber, BF, Scheuer, T and Catterall,
WA (1995) Structure and function of the beta 2 subunit of brain sodium channels, a
transmembrane glycoprotein with a CAM motif. Cell 83: 433-42.
Jackson, FR, Wilson, SD and Hall, LM (1986) The tip-E mutation of Drosophila decreases
saxitoxin binding and interacts with other mutations affecting nerve membrane
excitability. J Neurogenet 3: 1-17.
Jones, CM, Liyanapathirana, M, Agossa, FR, Weetman, D, Ranson, H, Donnelly, MJ and
Wilding, CS (2012) Footprints of positive selection associated with a mutation (N1575Y)
in the voltage-gated sodium channel of Anopheles gambiae. Proc Natl Acad Sci 109:
6614-6619.
Kasai, S, Ng, LC, Lam-Phua, SG, Tang, CS, Itokawa, K, Komogata, O, Kobayashi, M and
Tomita, T (2011) First detection of a putative knockdown resistance gene in major
mosquito vector, Aedes albopictus. Jpn J Infect Dis 64: 217-221.
Kawada, H, Higa, Y, Komagata, O, Kasai, S, Tomita, T, Yen, NT, Loan, LL, Sánchez, RAP and
Takagi, M (2009) Widespread distribution of a newly found point mutation in voltagegated sodium channel in pyrethroid-resistant Aedes aegypti populations in Vietnam. PLoS
Negl Trop Dis 3: e0000527.

157

Kim, HJ, Hawthorne, DJ, Peters, T, Dively, GP and Clark, JM (2005) Application of DNA-based
genotyping techniques for the detection of kdr-like pyrethroid resistance in field
populations of Colorado potato beetle. Pestic Biochem Physiol 81: 85-96.
Kiss, T (2008) Persistent Na-channels: origin and function. A review. Acta Biol Hung 59 Suppl:
1-12.
Kole, MH (2011) First node of Ranvier facilitates high-frequency burst encoding. Neuron 71:
671-82.
Kontis, KJ, Rounaghi, A and Goldin, AL (1997) Sodium channel activation gating is affected by
substitutions of voltage sensor positive charges in all four domains. J Gen Physiol 110:
391-401.
Kulkarni, SJ and Padhye, A (1982) Temperature-sensitive paralytic mutations on the second and
third chromosomes of Drosophila melanogaster. Genet Res 40: 191-199.
Kuruma, A and Hartzell, HC (1999) Dynamics of calcium regulation of chloride currents in
Xenopus oocytes. Am J Physiol 276: C161-75.
Lapied, B, Stankiewicz, M, Grolleau, F, Rochat, H, Zlotkin, E and Pelhate, M (1999)
Biophysical properties of scorpion alpha-toxin-sensitive background sodium channel
contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons). Eur
J Neurosci 11: 1449-60.
Lawrence, LJ and Casida, JE (1982) Pyrethroid toxicology: mouse intracerebral structuretoxicity relationships. Pestic Biochem Physiol 18: 9-14.
Le Corronc, H, Hue, B and Pitman, RM (1999) Ionic mechanisms underlying depolarizing
responses of an identified insect motor neuron to short periods of hypoxia. J
Neurophysiol 81: 307-18.
Lee, SH, Ingles, PJ, Knipple, DC and Soderlund, DM (2002) Developmental regulation of
alternative exon usage in the house fly Vssc1 sodium channel gene. Invert Neurosci 4:
125-133.
Lee, SH, Smith, TJ, Knipple, DC and Soderlund, DM (1999) Mutations in the house fly Vssc1
sodium channel gene associated with super-kdr resistance abolish the pyrethroid
sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes. Insect Biochem
Mol Biol 29: 185-194.
Li, Y and Bennett, DJ (2003) Persistent sodium and calcium currents cause plateau potentials in
motoneurons of chronic spinal rats. J Neurophysiol 90: 857-69.
Li, Y, Gorassini, MA and Bennett, DJ (2004) Role of persistent sodium and calcium currents in
motoneuron firing and spasticity in chronic spinal rats. J Neurophysiol 91: 767-83.
158

Lin, WH, Wright, DE, Muraro, NI and Baines, RA (2009) Alternative splicing in the voltagegated sodium channel DmNav regulates activation, inactivation, and persistent current. J
Neurophysiol 102: 1994-2006.
Littleton, JT and Ganetzky, B (2000) Ion channels and synaptic organization: analysis of the
Drosophila genome. Neuron 26: 35-43.
Liu, Z, Chung, I and Dong, K (2001) Alternative splicing of the BSC1 gene generates tissuespecific isoforms in the German cockroach. Insect Biochem Mol Biol 31: 703-13.
Liu, Z, Song, W and Dong, K (2004) Persistent tetrodotoxin-sensitive sodium current resulting
from U-to-C RNA editing of an insect sodium channel. Proc Natl Acad Sci 101: 1186211867.
Liu, Z, Tan, J, Huang, Z and Dong, K (2006) Effect of a fluvalinate-resistance-associated sodium
channel mutation from varroa mites on cockroach sodium channel sensitivity to
fulvalinate, a pyrethroid insecticide. Insect Biochem Mol Biol 36: 885-889.
Liu, Z, Tan, J, Valles, SM and Dong, K (2002) Synergistic interaction between two cockroach
sodium channel mutations and a tobacco budworm sodium channel mutation in reducing
channel sensitivity to a pyrethroid insecticide. Insect Biochem Mol Biol 32: 397-404.
Lombet, A, Mourre, C and Lazdunski, M (1988) Interaction of insecticides of the pyrethroid
family with specific binding sites on the voltage-dependent sodium channel from
mammalian brain. Brain Res 459: 44-53.
Loughney, K, Kreber, R and Ganetzky, B (1989) Molecular analysis of the para locus, a sodium
channel gene in Drosophila. Cell 58: 1143-1154.
Malhotra, JD, Kazen-Gillespie, K, Hortsch, M and Isom, LL (2000) Sodium channel beta
subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell
contact. J Biol Chem 275: 11383-8.
Mannikko, R, Elinder, F and Larsson, HP (2002) Voltage-sensing mechanism is conserved
among ion channels gated by opposite voltages. Nature 419: 837-41.
Mansotte, F, Ravachol, F, Carlisi, R, Caudal, J, Pinchon, S and Maison, D (2010) Operation to
promote use of long-lasting insecticidal nets (LLIN) in French Guiana in 2006: design,
implementation and results. Med Trop (Mars) 70: 249-54.
Marley, R and Baines, RA (2011) Increased persistent Na+ current contributes to seizure in the
slamdance bang-sensitive Drosophila mutant. J Neurophysiol 106: 18-29.
Martinez-Torres, D, Chandre, F, Williamson, MS, Darriet, F, Berge, JB, Devonshire, AL,
Guillet, P, Pasteur, N and Pauron, D (1998) Molecular characterization of pyrethroid
159

knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect
Mol Biol 7: 179-84.
McCormick, KA, Isom, LL, Ragsdale, D, Smith, D, Scheuer, T and Catterall, WA (1998)
Molecular determinants of Na+ channel function in the extracellular domain of the beta1
subunit. J Biol Chem 273: 3954-62.
Meadows, LS, Chen, YH, Powell, AJ, Clare, JJ and Ragsdale, DS (2002) Functional modulation
of human brain Nav 1.3 sodium channels, expressed in mammalian cells, by auxiliary
beta1, beta2 and beta3 subunits. Neuroscience 114: 745-753.
Meisler, MH and Kearney, JA (2005) Sodium channel mutations in epilepsy and other
neurological disorders. J Clin Invest 115: 2010-7.
Mitrovic, N, George, AL, Jr. and Horn, R (2000) Role of domain 4 in sodium channel slow
inactivation. J Gen Physiol 115: 707-18.
Moran, Y, Kahn, R, Cohen, L, Gur, M, Karbat, I, Gordon, D and Gurevitz, M (2007) Molecular
analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the
heterogeneity of receptor site-3 on voltage-gated Na+ channels. Biochem J 15;406(1):418.
Morgan, K, Stevens, EB, Shah, B, Cox, PJ, Dixon, AK, Lee, K, Pinnock, RD, Hughes, J,
Richardson, PJ, Mizuguchi, K and Jackson, AP (2000) beta 3: an additional auxiliary
subunit of the voltage-sensitive sodium channel that modulates channel gating with
distinct kinetics. Proc Natl Acad Sci 97: 2308-13.
Motomura, H and Narahashi, T (2000) Temperature dependence of pyrethroid modification of
single sodium channels in rat hippocampal neurons. J Membr Biol 177: 23-39.
Narahashi, T (1986) Nerve membrane ionic channels as the target of toxicants. Arch Toxicol
Suppl 9: 3-13.
Narahashi, T (1987) Neuronal Target Sites of Insecticides. In: Sites of Action for Neurotoxic
Pesticides (Hollingworth, RM and Green, MB, eds.). Vol. 356, pp. 226-250. ACS
Symposium Series.
Narahashi, T (1992) Nerve Membrane Na+ Channels as Targets of Insecticides. TiPS Rev. 13:
236-241.
Narahashi, T (1996) Neuronal ion channels as the target sites of insecticides. Pharmacol Toxicol
78: 1-14.
Narahashi, T (2000) Neuroreceptors and ion channels as the basis for drug action: past, present,
and future. J Pharmacol Exp Ther 294: 1-26.

160

Noda, M, Shimizu, S, Tanabe, T, Takai, T, Kayano, T, Ikeda, T, Takahashi, H, Nakayama, H,
Kanaoka, Y, Minamino, KK, Kangawa, L, Matsuo, H, Raftery, MA, Hirose, TISHH and
Miyata, TaNS (1984) Primary Structure of Electrophorus Electricus Sodium Channel
Deduced from cDNA Sequence. Nature 312: 121-127.
O'Donnell-Olson, R, Liu, Z, Nomura, Y, Song, W and Dong, K (2008) Molecular and functional
characterization of voltage-gated sodium channel variants from Drosophila
melanogaster. Insect Biochem Mol Biol 38: 604-610.
O'Dowd, DK (1995) Voltage-gated currents and firing properties of embryonic Drosophila
neurons grown in a chemically defined medium. J Neurobiol 27: 113-26.
O'Dowd, DK and Aldrich, RW (1988) Voltage-clamp analysis of sodium channels in wild-type
and mutant Drosophila neurons. J Neurosci 8: 3633-43.
O'Reilly, AO, Khambay, BPS, Williamson, MS, Field, LA, Wallace, BA and Davies, TGE
(2006) Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem
J 396: 255-263.
Oh, Y, Sashihara, S, Black, JA and Waxman, SG (1995) Na+ channel beta 1 subunit mRNA:
differential expression in rat spinal sensory neurons. Brain Res Mol Brain Res 30: 35761.
Oliveira, EE, Du, Y, Nomura, Y and Dong, K (2013) A residue in the transmembrane segment 6
of domain I in insect and mammalian sodium channels regulate differential sensitivities
to pyrethroid insecticides. Neurotoxicology 38C: 42-50.
Olson, RO, Liu, Z, Nomura, Y, Song, W and Dong, K (2008) Molecular and functional
characterization of voltage-gated sodium channel variants from Drosophila melanogaster.
Insect Biochem Mol Biol 38: 604-10.
Palladino, MJ, Keegan, LP, O'Connell, MA and Reenan, RA (2000) A-to-I pre-mRNA editing in
Drosophila is primarily involved in adult nervous system function and integrity. Cell
102: 437-449.
Patino, GA and Isom, LL (2010) Electrophysiology and beyond: multiple roles of Na+ channel
beta subunits in development and disease. Neurosci Lett 486: 53-9.
Pauron, D, Barhanin, J, Amichot, M, Pralavorio, M, Berge, JB and Lazdunski, M (1989)
Pyrethroid Receptor in the Insect Na+ Channel: Alteration of Its Properties in PyrethroidResistant Flies. Biochem 28: 1673-1377.
Pennartz, CM, Bierlaagh, MA and Geurtsen, AM (1997) Cellular mechanisms underlying
spontaneous firing in rat suprachiasmatic nucleus: involvement of a slowly inactivating
component of sodium current. J Neurophysiol 78: 1811-25.

161

Qu, Y, Curtis, R, Lawson, D, Gilbride, K, Ge, P, DiStefano, PS, Silos-Santiago, I, Catterall, WA
and Scheuer, T (2001) Differential modulation of sodium channel gating and persistent
sodium currents by the beta1, beta2, and beta3 subunits. Mol Cell Neurosci 18: 570-80.
Rakowski, RF, Gadsby, DC and De Weer, P (2002) Single ion occupancy and steady-state gating
of Na channels in squid giant axon. J Gen Physiol 119: 235-49.
Raman, IM and Bean, BP (1997) Resurgent sodium current and action potential formation in
dissociated cerebellar Purkinje neurons. J Neurosci 17: 4517-26.
Ranson, H, Jensen, B, Vulule, JM, Wang, X, Hemingway, J and Collins, FH (2000)
Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan
Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol 9:
491-497.
Reenan, RA, Hanrahan, CJ and Ganetzky, B (2000) The mle(napts) RNA helicase mutation in
drosophila results in a splicing catastrophe of the para Na+ channel transcript in a region
of RNA editing. Neuron 25: 139-49.
Rinkevich, FD, Du, Y and Dong, K (2013) Diversity and Convergence of Sodium Channel
Mutations Involved in Resistance to Pyrethroids. Pestic Biochem Physiol 106: 93-100.
Rohl, CA, Boeckman, FA, Baker, C, Scheuer, T, Catterall, WA and Klevit, RE (1999) Solution
structure of the sodium channel inactivation gate. Biochem 38: 855-61.
Rossignol, DP (1988) Reduction in Number of Nerve Membrane Sodium Channels in Pyrethroid
Resistant House Flies. Pestic Biochem Physiol 32: 146-152.
Saavedra-Rodriguez, K, Urdaneta-Marquez, L, Rajatileka, S, Moulton, M, Flores, AE,
Fernandez-Salas, I, Bisset, J, Rodriguez, M, Mccall, PJ, Donnelly, MJ, Ranson, H,
Hemingway, J and Black, WCI (2007) A mutation in the voltage-gated sodium channel
gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol
Biol 16: 785-798.
Saito, M and Wu, CF (1993) Ionic channels in cultured Drosophila neurons. EXS 63: 366-89.
Schafer, S, Rosenboom, H and Menzel, R (1994) Ionic currents of Kenyon cells from the
mushroom body of the honeybee. J Neurosci 14: 4600-12.
Singh, OP, Dykes, CL, Das, MK, Pradhan, S, Bhatt, RM, Agrawal, OP and Adak, T (2010)
Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel
mutation V1010L, in the voltage-gated Na+ channel of Anopheles culicifacies from
Orissa, India. Malaria J 9: 146.

162

Smith, TJ, Lee, SH, Ingles, PJ, Knipple, DC and Soderlund, DM (1997) The L1014F point
mutation in the house fly Vssc1 sodium channel confers knockdown resistance to
pyrethroids. Insect Biochem Mol Biol 27: 807-812.
Soderlund, D, M. , Clark, JM, Sheets, LP, Mullin, LS, Piccirillo, VJ, Sargent, D, Stevens, JT and
Weiner, ML (2002) Mechanisms of pyrethroid neurotoxicity: Implications for cumulative
risk assessment. Toxicol Appl Pharmacol 171: 3-59.
Soderlund, DM (2005) Sodium channels. In: Comprehensive molecular insect science (Gilbert,
LI, Iatrou, K and Gill, SS, eds.). Vol. 5, pp. 1-24. Elsevier, Amsterdam.
Soderlund, DM (2008) Pyrethroids, knockdown resistance and sodium channels. Pest Man Sci
64: 610-616.
Soderlund, DM (2012) Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent
advances. Arch Toxicol 86: 165-181.
Soderlund, DM and Bloomquist, JR (1989) Neurotoxic actions of pyrethroid insecticides. Ann
Rev Entomol 34: 77-96.
Soderlund, DM and Bloomquist, JR (1990) Molecular Mechanisms of Insecticide Resistance. In:
Pesticide Resistance in Arthropods (Roush, RT and Tabashnik, BE, eds.). pp. 58.
Chapman and Hall, New York and London.
Song, J-H and Narahashi, T (1996) Modulation of sodium channels of rat cerebellar Purkinje
neurons by the pyrethroid tetramethrin. J Pharmacol Exper Therap 277: 445-453.
Song, W, Liu, Z and Dong, K (2006) Molecular basis of differential sensitivity of insect sodium
channels to DCJW, a bioactive metabolite of the oxadiazine insecticide indoxacarb.
NeuroToxicology 27: 237-244.
Song, W, Liu, Z, Tan, J, Nomura, Y and Dong, K (2004) RNA editing generates tissue-specific
sodium channels with distinct gating properties. J Biol Chem 279: 32554-32561.
Suzuki, DT, Grigliatti, T and Williamson, R (1971) Temperature-sensitive mutations in
Drosophila melanogaster, VII. A mutation (parats) causing reversible adult paralysis.
Proc Natl Acad Sci 68: 890-893.
Syafruddin, D, Hidayati, AP, Asih, PB, Hawley, WA, Sukowati, S and Lobo, NF (2010)
Detection of 1014F kdr mutation in four major Anopheline malaria vectors in Indonesia.
Malar J 9: 315.
Takahashi, N, Kikuchi, S, Dai, Y, Kobayashi, K, Fukuoka, T and Noguchi, K (2003) Expression
of auxiliary beta subunits of sodium channels in primary afferent neurons and the effect
of nerve injury. Neuroscience 121: 441-50.

163

Takeda, K and Narahashi, T (1988) Chemical modification of sodium channel inactivation:
separate sites for the action of grayanotoxin and tetramethrin. Brain Res 448: 308-312.
Tan, J, Liu, Z, Tsai, TD, Valles, SM, Goldin, AL and Dong, K (2002a) Novel sodium channel
gene mutations in Blattella germanica reduce the sensitivity of expressed channels to
deltamethrin. Insect Biochem Mol Biol 32: 445-454.
Tan, J, Liu, Z, Wang, R, Huang, ZY, Chen, AC, Gurevitz, M and Dong, K (2005) Identification
of amino acid residues in the insect sodium channel critical for pyrethroid binding. Mol
Pharmacol 67: 513-22.
Tan, J and Soderlund, DM (2011) Independent and joint modulation of rat Nav1.6 voltage-gated
sodium channels by coexpression with the auxiliary beta1 and beta2 subunits. Biochem
Biophys Res Commun 407: 788-92.
Tan, J, Z. Liu, Z, Nomura, Y, Goldin, AL and Dong, K (2002b) Alternative splicing of an insect
sodium channel gene generates pharmacologically distinct sodium channels. J Neurosci
22: 5300-5309.
Tan, WL, Li, CX, Wang, ZM, Liu, MD, Dong, YD, Feng, XY, Wu, ZM, Guo, XX, Xing, D,
Zhang, YM, Wang, ZC and Zhao, TY (2012) First detection of multiple knockdown
resistance (kdr)-like mutations in voltage-gated sodium channel using three new
genotyping methods in Anopheles sinensis from Guangxi Province, China. J Med
Entomol 49: 1012-1020.
Tatebayashi, H and Narahashi, T (1994) Differential mechanism of action of the pyrethroid
tetramethrin on tetradotoxin-sensitive and tetradotoxin-resistant sodium channels. J
Pharmacol Exper Therap 270: 595-603.
Terlau, H, Heinemann, SH, Stuhmer, W, Pusch, M, Conti, F, Imoto, K and Numa, S (1991)
Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett
293: 93-6.
Thackeray, JR and Ganetzky, B (1994) Developmentally regulated alternative splicing generates
a complex array of Drosophila para sodium channel isoforms. J Neurosci 14: 2569-78.
Thackeray, JR and Ganetzky, B (1995) Conserved alternative splicing patterns and splicing
signals in the Drosophila sodium channel gene para. Genetics 141: 203-14.
Unkiewicz-Winiarczyk, A and Gromysz-Kalkowska, K (2012) Effect of temperature on toxicity
of deltamethrin and oxygen consumption by Porcellio scaber Latr (Isopoda). Bull Environ
Contam Toxicol 89: 960-5.
Vais, H, S. Atkinson, N. Eldursi, A.L. Devonshire, M.S. Williamson, and P.N.R. Usherwood
(2000) A single amino acid change makes a rat neuronal sodium channel highly sensitive
to pyrethroid insecticides. FEBS Letters 470: 135-138.
164

Vais, H, Williamson, MS, Goodson, SJ, Devonshire, AL, Warmke, JW, Usherwood, PNR and
Cohen, CJ (2000) Activation of Drosophila sodium channels promotes modification by
deltamethrin: Reductions in affinity caused by knock-down resistance mutations. J of
Gen Physiol 115: 305-318.
Verhaeghen, K, van Bortel, W, Trung, HD, Sochantha, T, Keokenchanh, K and Coosemans, M
(2010) Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An.
peditaeniatus populations of the Mekong region. Parasit Vect 3: 59.
Vijayaragavan, K, O'Leary, ME and Chahine, M (2001) Gating properties of Na(v)1.7 and
Na(v)1.8 peripheral nerve sodium channels. J Neurosci. 21: 7909-18.
Vijayaragavan, K, Powell, AJ, Kinghorn, IJ and Chahine, M (2004) Role of auxiliary beta1-,
beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium
channel. Biochem Biophys Res Commun 319: 531-40.
Vijverberg, HP, van der Zalm, JM and van der Bercken, J (1982) Similar mode of action of
pyrethroids and DDT on sodium channel gating in myelinated nerves. Nature 295: 601-3.
Vijverberg, HPM and van den Bercken, J (1990) Neurotoxicological effects and the mode of
action of pyrethroid insecticides. Crit Rev Toxicol 21: 105-126.
Vilin, YY, Makita, N, George Jr, AL and Ruben, PC (1999) Structural determinants of slow
inactivation in human cardiac and skeletal muscle sodium channels. Biophys J 77: 13841393.
Vilin, YY and Ruben, PC (2001) Slow inactivation in voltage-gated sodium channels: molecular
substrates and contributions to channelopathies. Cell Biochem Biophys 35: 171-90.
Wang, J, Yarov-Yarovoy, V, Kahn, R, Gordon, D, Gurevitz, M, Scheuer, T and Catterall, WA
(2011) Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage
sensor. Proc Natl Acad Sci 108: 15426-31.
Wang, L, Nomura, Y, Du, Y and Dong, K (2013) Differential effects of TipE and a TipEhomologous protein on modulation of gating properties of sodium channels from
Drosophila melanogaster. Plos One 8: e67551.
Warmke, JW, Reenan, RAG, Wang, P, Qian, S, Arena, JP, Wang, J, Wunderler, D, Liu, K,
Kaczorowski, GJ, Van Der Ploeg, LHT, Ganetzky, B and Cohen, CJ (1997) Functional
expression of Drosophila para sodium channels. J Gen Physiol 110: 119-133.
Webb, J, Wu, FF and Cannon, SC (2009) Slow inactivation of the Nav1.4 sodium channel in
mammalian cells is impeded by co-expression of the beta1 subunit. Pflugers Arch 457:
1253-63.

165

Wicher, D, Walther, C and Wicher, C (2001) Non-synaptic ion channels in insects--basic
properties of currents and their modulation in neurons and skeletal muscles. Prog
Neurobiol 64: 431-525.
Willey, BA, Paintain, LS, Mangham, L, Car, J and Schellenberg, JA (2012) Strategies for
delivering insecticide-treated nets at scale for malaria control: a systematic review. Bull
World Health Organ 90: 672-684E.
Xu, R, Thomas, EA, Gazina, EV, Richards, KL, Quick, M, Wallace, RH, Harkin, LA, Heron,
SE, Berkovic, SF, Scheffer, IE, Mulley, JC and Petrou, S (2007) Generalized epilepsy
with febrile seizures plus-associated sodium channel beta1 subunit mutations severely
reduce beta subunit-mediated modulation of sodium channel function. Neuroscience.
148: 164-74.
Yu, FH and Catterall, WA (2003) Overview of the voltage-gated sodium channel family.
Genome Biol 4: 207.
Yu, FH, Westenbroek, RE, Silos-Santiago, I, McCormick, KA, Lawson, D, Ge, P, Ferriera, H,
Lilly, J, DiStefano, PS, Catterall, WA, Scheuer, T and Curtis, R (2003) Sodium channel
beta4, a new disulfide-linked auxiliary subunit with similarity to beta2. J Neurosci 23:
7577-85.
Zhang, T, Liu, Z, Song, W, Du, Y and Dong, K (2011) Molecular characterization and functional
expression of the DSC1 channel. Insect Biochem Mol Biol 41: 451-8.
Zhao, J, O'Leary, ME and Chahine, M (2011) Regulation of Nav1.6 and Nav1.8 peripheral nerve
Na+ channels by auxiliary beta-subunits. J Neurophysiol 106: 608-19.
Zhao, Y, Park, Y and Adams, ME (2000) Functional and evolutionary consequences of
pyrethroid resistance mutations in S6 transmembrane segments of a voltage-gated sodium
channel. Biochem Biophys Res Commun 278: 516-521.

166